Tagged oliggonucleotides and their use in nucleic acid amplification methods

[0001] This invention relates to methods, compositions, reaction mixtures and kits for the selective amplification of multiple copies of a specific nucleic acid sequence or "target sequence" which may be present either alone or as part of a homogeneous or heterogeneous mixture of nucleic acids. A mixture of nucleic acids can be that found in a sample taken for diagnostic tests, screening for blood products, sterility tests, microbiological detection in food, water, beverages, industrial or environmental samples, scientific studies, preparation of reagents or materials for other processes such as cloning or other purposes. The selective amplification of specific nucleic acid sequences as described herein is of particular importance in any of a variety of detection tests to increase the accuracy and reliability of such tests, while reducing the requirements for the production, purification and / or sterilization of the reagents used in these tests and the test environment. are being done.

DESCRIPTION OF RELATED FIELD [0002] Detection and / or quantification of specific nucleic acid sequences is an important technique for identifying and classifying microorganisms, diagnosing infectious diseases, measuring responses to various types of therapies, and the like. Such procedures are also useful in the detection and quantification of microorganisms in foodstuffs, water, beverages, industrial and environmental samples, seed stocks and other types of materials where monitoring of the presence of specific microorganisms may be desirable.

[0003] Numerous amplification-based methods for the detection and quantification of target nucleic acids are well known and defined in the art. Polymerase chain reactions, commonly known as PCR, use multiple denaturation cycles, attachment of primer pairs to opposite strands, and primer extension to exponentially increase the number of copies of the target sequence (e.g. Mullis et al., "Process for Amplifying, Detecting and / or Cloning Nucleic Acid Sequences", US Patent No. 4,683195; Mullis, "Process for Amplifying Nucleic Acid Sequences", US Patent No. 4,683,202; Mullis et al., "Process for Amplifying, Detecting and / or Cloning Nucleic Acid

Sequences', US Patent No. 4,800-159; Gelfand et al., "Reaction Mixtures for the Detection of Target Nucleic Acids", US Patent No. 5,804375; Mullis et al. (1987) Meth. Enzymol. 155, 335-350; and Murakawa et al. (1988) DNA 7.287-295).

[0004] In a variation called RT-PCR, reverse transcriptase (RT) is used to produce complementary DNA (cDNA) from RNA, and then the cDNA is amplified by PCR to generate multiple copies of DNA (Gelfand et al., "Reverse Transcription with Thermostable DNA Polymerases - High Temperature Reverse Transcription ", US Patent Nos. 5,322770 and 5310652).

[0005] Another well known method of amplification is strand displacement amplification, commonly referred to as SDA, which uses cycles to attach primer sequence pairs to opposite strands of the target sequence, primer extension in the presence of dNTP to form a double-stranded hemithiophosphorylated primer extension product, mediated by endonuclease cleavage hemi-modified restriction endonuclease recognition sites, and polymerase-mediated primer extension from the 3'-end of the incision to displace the existing strand and create the strand for the next round of primer attachment, strand cutting and displacement, resulting in geometric product amplification (e.g. Walker, G. et al. (1992), Proc. Natl. Acad. Sci. USA 89, 392-396; Walker et al., "Nucleic Acid Target Generation", US Patent No. 5,272,184; Walker, "Strand Displacment Amplification", US Patent No. 5,455,166; and Walker and in. (1992) Nucleic Acids Research 20, 1691-1696). Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same way (European Patent No. 0 684 315).

[0006] Other amplification methods include "rolling circle" (RCA) amplification (eg, Lizardi, "Rolling Circle Replication Reporter Systems", US Patent No. 5,854033); helicase-dependent (HDA) amplification (e.g. Kong et al., "Helicase Dependent Amplification Nucleic Acids", US Patent Application Publication No. US 20040058378 A1); and isothermal loop-mediated amplification (LAMP) (e.g., Notomi et al., "Process for Synthesizing Nucleic Acid", US Patent No. 6,410278).

[0007] Transcription-based amplification methods commonly used in the art include nucleic acid sequence-based amplification, also called NASBA (e.g., Malek et al., US Patent No. 5,130,238); methods that rely on the use of RNA replicase to amplify the probe molecules themselves, commonly referred to as Qe replicase (e.g., Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202); transcription based amplification methods (e.g., Kwoh, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86,

1173-1177) and self-persistent sequence replication (e.g., Guatelli, J. et al. (1990) Proc.

Natl. Acad. Sci. USA 87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; and HELEN H. LEE et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)). [0008] Another method of transcription-based amplification is transcription-mediated amplification, commonly called TMA, which autocatalytically synthesizes multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH in which multiple copies of the target sequence RNA autocatalytically produce additional copies (eg. Kacian et al., "Nucleic Acid Sequence Amplification Methods", US Patent No. 5,480,784; and Kacian et al., U.S. Patent No. 5,399,491). TMA is a strong and very sensitive amplification system with demonstrated efficacy, which overcomes many of the problems associated with PCR-based amplification systems. In particular, no cyclic temperature changes are required.

[0009] Amplification tests are particularly well suited for the detection of microorganisms in the context of clinical laboratory tests, monitoring of biological processes or in any other conditions in which it is desirable to detect microorganisms in a particular type of sample, due to the fact that they provide high sensitivity and quick time to obtain a result in compared with standard microbiological techniques. In addition, amplification methods can be used to detect a large number of microorganisms that are difficult or impossible to grow on artificial media. However, there are some limitations associated with first generation amplification tests that have limited their use at specific locations, such as clinical microbiology laboratories. One inherent problem associated with the high sensitivity of nucleic acid amplification systems is that the contaminating nucleic acid introduced into the amplification system (e.g., from one or more reagents used during the amplification, from the test technician, from the environment in which the amplification is performed, etc. ) may give false positive results. For example, even unusually small amounts of nucleic acid impurities present in reagents and / or enzymes used in the amplification reaction or in the environment in which the amplification reaction is carried out can lead to a positive signal of amplification, although the sequence of interest is not present in the nucleic acid sample tested. This requires some effort in sample preparation, cleaning, sterilization, etc. reagents used in amplification reactions to avoid or minimize false positive results.

[0010] Accordingly, there remains a need in the art for a strong nucleic acid amplification system that can selectively amplify one or more nucleic acid target sequences of interest, while reducing or eliminating false positive results that may occur as a result of the presence of contaminating biological material, such as a contaminating nucleic acid. There is also a need for amplification systems with reduced requirements for reagent purity and / or sterility. As further described herein, the present invention responds to these needs and provides other related benefits.

SUMMARY OF THE INVENTION [0011] The present invention generally relates to nucleic acid amplification methods that desirably limit or eliminate false positive amplification signals resulting from contaminating biological material, e.g., nucleic acid, which may be present in one or more reagents, components or materials that are used in the amplification reaction or are present in the environment in which the amplification reaction is carried out. The invention further provides the advantage of requiring less stringent purification and / or sterility procedures than those normally required to ensure that enzymes and other reagents and components used in amplification reactions are free of bacteria and other nucleic acid contaminants that may give false positive results. Such ingredients or materials include, but are not limited to, water, buffers, salts, solid substrates (e.g., magnetically charged particles or spheres), and vessels (e.g., glass or plastic). Accordingly, the methods of the invention are useful in the detection and / or quantification of microorganisms in clinical samples, foodstuffs, water, industrial and environmental samples, seed stocks and other types of materials where there may be a need to detect and / or monitor the presence of microorganisms. The methods of the invention have particular advantages for testing raw materials used to manufacture products for the biotechnology, pharmaceutical, cosmetics and beverage industries, final product release tests and sterility screening to test for the presence of a certain class of organisms or all living organisms in the material of interest (bacteria) , mushrooms or both). In clinical conditions, the methods of the invention will be particularly useful for testing sepsis, especially blood poisoning, which is caused by pathogenic organisms and / or their toxins in the bloodstream. The methods of the invention are defined in the claims.

[0012] According to one embodiment of the present disclosure, methods are provided for selectively amplifying at least one target nucleic acid sequence, such as a DNA or RNA sequence, which method comprises the steps of: (a) acting on a target nucleic acid sequence in a nucleic acid sample, for example. wherein the target nucleic acid is immobilized on a solid support by a heterologous tag sequence to form a tagged target nucleic acid sequence; (b) reducing in this sample the effective concentration of heterologous tag sequences that have not become part of this tagged target nucleic acid sequence and exist in a form capable of producing a tagged target nucleic acid sequence with that target nucleic acid sequence; and (c) exposing said labeled target nucleic acid sequence to reagents and conditions sufficient for detectably amplifying the target nucleic acid sequence, wherein at this stage of exposure the nucleic acid sample is exposed to a known contaminant source of the target nucleic acid sequence after step (b) and wherein the detectable amplification of the target nucleic acid sequence is essentially limited to the amplification of the target nucleic acid sequence provided in the tagged target nucleic acid sequence from step (a) and not the target nucleic acid sequence provided from a known contaminant source.

[0013] The methods of the disclosure are particularly useful when one or more of the reagents or components used are made from a material known as a contaminant source of the amplified target nucleic acid sequence. In one example, one or more reagents used in the methods, such as nucleic acid polymerases, are prepared using a microorganism containing the target nucleic acid sequence. In another example, the components used in the methods, such as reaction vessels, pipette tips, and solid supports for binding to labeled target nucleic acid sequences may be a known contaminant source of the target nucleic acid sequence. In addition, these methods are useful when the environmental conditions in which the amplification is performed include a known source of contaminating the target nucleic acid sequence, such as ambient air, contractor and analytical apparatus.

[0014] In a more particular aspect of this embodiment, during step (b) the tagged target nucleic acid sequence is immobilized on a solid support.

[0015] In another particular aspect, step (b) comprises diluting or removing heterologous tag sequences that have not become part of the tagged target nucleic acid sequence from the nucleic acid sample. In an alternative aspect, step (b) comprises inactivating heterologous tag sequences that have not become part of this tagged target nucleic acid sequence to form an inactivated heterologous tag sequence. In a related aspect, the method further comprises removing the inactivated heterologous tag sequence from this nucleic acid sample during step (b). The heterologous tag sequence can be inactivated by blocking its ability to form a complex with the target nucleic acid sequence, using an enzyme to digest the component or to cleave the site of a portion of the heterologous tag sequence forming the complex, chemical alteration of the heterologous tag sequence or change in the ability of the heterologous tag sequence to form a complex with the target nucleic acid sequence in the reaction mixture for amplification by other means.

[0016] In another aspect, the heterologous tag sequence is contained in a tagged oligonucleotide, which tagged oligonucleotide comprises a first and a second region, wherein the first region comprises a target hybridizing sequence that hybridizes to the 3'-end of the target nucleic acid sequence, and wherein the second region comprises a tag sequence located 5 'to the target hybridizing sequence, and wherein the tag sequence does not stably hybridize to the target nucleic acid containing the target nucleic acid sequence.

[0017] In yet another aspect, the heterologous tag sequence during step (a) is in active form, which allows the heterologous tag sequence to produce a tagged target nucleic acid sequence, and wherein in step (b) the heterologous tag sequence that did not produce the tagged target sequence nucleic acid is transformed into an inactive form, which blocks the production of the tagged target nucleic acid sequence by the heterologous tag sequence during step (c).

[0018] In certain aspects, the target hybridizing sequence is a universal oligonucleotide, such as a universal bacterial or fungal oligonucleotide.

[0019] Step (c) comprises producing amplification products in a nucleic acid amplification reaction using the first and second oligonucleotides, wherein the first oligonucleotide comprises a sequence that hybridizes to the 3'-end of the complement of the target nucleic acid sequence, and wherein the second oligonucleotide comprises the sequence which hybridizes to the complement of the tag sequence, but which does not stably hybridize to the target nucleic acid sequence, wherein each of the amplification products comprises a base sequence that is substantially identical or complementary to the base sequence of the target nucleic acid sequence and further comprises a base sequence that is substantially identical or complementary to all or a portion of the tag sequence.

[0020] Various amplification methods are suitable for use in the present invention. For example, in one aspect, the amplification reaction is a PCR reaction. In another aspect, the target nucleic acid sequence is amplified by a transcription based amplification reaction, preferably a TMA reaction carried out under isothermal conditions.

[0021] The target nucleic acid sequence amplified according to these methods may be any target sequence of interest, but will typically be a nucleic acid sequence obtained from a microorganism. Furthermore, the method can be selective for the amplification of the target nucleic acid sequence contained in the nucleic acid of a single strain or species of microorganisms or in many species of microorganisms. Alternatively, the method may be selective for the amplification of multiple target nucleic acid sequences contained in the nucleic acid of many species of microorganisms, wherein, for example, in step (a) the target hybridizing sequence from the tagged oligonucleotide hybridizes to the target region present in each of the multiple target nucleic acid sequences .

[0022] For example, in a particular aspect, the method is selective for the amplification of a target nucleic acid sequence contained in each of a plurality of target nucleic acids, and one wherein in step (a) the heterologous tag sequence produces a tagged target nucleic acid sequence with the target nucleic acid sequence from each of the numerous target nucleic acids present in the nucleic acid sample. In a more particular aspect, the target nucleic acid sequence contained in each of the plurality of target nucleic acids is the same nucleic acid sequence.

[0023] In another particular aspect, the method is selective for the amplification of multiple bacterial or fungal target nucleic acid sequences, e.g., wherein the multiple bacterial or fungal target nucleic acid sequences are ribosomal nucleic acid sequences.

In another particular aspect, the method is selective for the amplification of target nucleic acid sequences obtained from organisms belonging to the group of bacterial species including Staphylococci species (e.g. Staphylococcus aureus, Staphylococcus epidermis and Staphylococcus haemolyticus), Steptococci species (e.g. Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci and beta-hemolytic streptococci), Enterococcus species (e.g. Enterococcus faecium and Enterococcus faecalis), Escherichia species (e.g. Escherichia Kielsla) Klebsiella oxytoca), Pseudomonas species (e.g. Pseudomonas aeruginosa), Enterobacter species (e.g. Enterobacter cloacae and Enterobacter aerogenes), Proteus species (e.g. Proteus mirabilis), Bacterioides species, species

Clostridium, Serratia species (e.g. Serratia marcescens), Acinetobaeter species (e.g. Acinetobacter baumannii) and Stenotrophomonas species (e.g. Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0025] In another aspect, the method is selective for the amplification of target nucleic acid sequences obtained from organisms belonging to the group of fungal species including Candida species (e.g., Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae; Candida krusel, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis and Candida famata), Histoplama capsulatum, Cryptococcus species (e.g. Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii), Coccidioides species (e.g. Coccidioides immitis), Trichosporon species (e.g. Trichosporon cutaneum), Malassezia species (e.g. Malassezia furfur), Rhodotorula species, Nocardia species (e.g. Nocardia species) Fusarium and Asperigillus species (e.g., Asperigillus fumigalus). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0026] In yet another aspect, at least a portion of the nucleic acid sample used in these methods is obtained from a clinical, water, industrial, environmental, seed, beverage or food source.

[0027] In certain aspects, these methods are particularly well suited for use in sterility tests or diagnostic tests for sepsis. [0028] According to another embodiment of the disclosure, there is provided a method of selectively amplifying at least one target nucleic acid sequence from a nucleic acid sample, the method comprising the steps of: (a) treating a nucleic acid sample comprising a target nucleic acid sequence labeled with an oligonucleotide comprising the first and second regions, wherein the first region comprises a target hybridizing sequence that hybridizes to the 3'-end of the target nucleic acid sequence and wherein the second region comprises the tag sequence located 5 'to the target hybridizing sequence, wherein the second region does not stably hybridize to the target nucleic acid containing the target nucleic acid sequence; (b) reducing in this nucleic acid sample the effective concentration of unhybridized tagged oligonucleotide in active form in which the target hybridizing sequence of that unhybridized tagged oligonucleotide is available for hybridization with that target nucleic acid sequence; and (c) producing amplification products in a nucleic acid amplification reaction using the first and second oligonucleotides, wherein the first oligonucleotide comprises a hybridizing sequence that hybridizes to the 3'-end of the complement of the target nucleic acid sequence and wherein the second oligonucleotide comprises a hybridizing sequence that hybridizes with marker sequence completion, wherein the second oligonucleotide hybridizes stably to the target nucleic acid sequence, and wherein each of the amplification products comprises a base sequence that is substantially identical or complementary to the base sequence of the target nucleic acid sequence and further comprises a base sequence that is substantially identical or complementary to all or part of the tag sequence.

[0029] In one aspect of the above methods during step (b), at least one target nucleic acid sequence is immobilized on a solid support. In another aspect, step (b) does not include the use of an enzyme with nuclease activity.

[0030] The effective concentration of unhybridized tagged oligonucleotide in active form is preferably reduced prior to amplification by diluting the nucleic acid sample or by inactivating and / or removing unhybridized tagged oligonucleotide. In one aspect, step (b) comprises inactivating an unhybridized tagged oligonuleotide, such that the unhybridized tagged oligonucleotide does not stably hybridize to the target nucleic acid sequence during step (c). In one example of inactivation, the tagged oligonucleotide during step (a) is in an active form that allows the target hybridizing sequence to hybridize to the target nucleic acid sequence, and wherein in step (b) the unhybridized tagged oligonucleotide is converted to the inactive form, which blocks or prevents hybridizing the tagged oligonucleotide to the target nucleic acid sequence during step (c). The tagged oligonucleotide can be inactivated by blocking the target hybridizing sequence prior to hybridization with the target nucleic acid sequence using an enzyme to digest the component or cleave the duplex site generated between the target hybridizing sequence and the target nucleic acid sequence, chemical alteration of the target hybridizing sequence or change in the ability of the tagged oligonucleotide to hybridize to the target nucleic acid sequence in the reaction mixture for amplification by other means.

[0031] In a related embodiment, the conditions in steps (b) and (c) are less stringent than the conditions in step (a). In another related embodiment, the temperature of the nucleic acid sample is lowered between steps (a) and (b).

[0032] In another example, where step (b) comprises inactivating the unhybridized tagged oligonucleotide, in step (b) the unhybridized tagged oligonucleotide of step (a) is converted from a single-stranded form into a duplex form. The duplex form may be a hairpin tag molecule comprising the tag closing sequence attached to the 5'-end of the tagged oligonucleotide, wherein the tag closing sequence hybridizes to the target hybridizing sequence under the conditions of step (b), thereby blocking hybridization of the unhybridized tagged oligonucleotide step (a) with the target nucleic acid sequence in steps (b) and (c). In another aspect, the tag closing sequence is attached to the tagged oligonucleotide via a non-nucleotide linker. For example, the 5'-end of the tag closing sequence may be attached to the 5'-end of the tagged oligonucleotide. [0033] The tagged oligonucleotide may also further comprise a third region containing a promoter for RNA polymerase, which third region is located 5 'to the second region.

[0034] In another aspect, the tag closing sequence is modified to prevent DNA synthesis from starting therefrom.

[0035] According to another aspect, the 3'-terminal base of the target hybridizing sequence is hybridized to the 5'-terminal base of the tag closing sequence. In another aspect, the 3'-end of the tag closing sequence is joined to the 5'-end of the tagged oligonucleotide.

[0036] In yet another aspect, in step (b) the target hybridizing sequence is hybridized to a tag closing oligonucleotide, wherein the tagged oligonucleotide and tag closing oligonucleotide are separate molecules. The tag closing oligonucleotide can be modified when desired to prevent it from starting DNA synthesis.

[0037] Furthermore, in certain aspects, the 3'-terminal base of the target hybridizing sequence is hybridized to the 5'-terminal base of the tag closing oligonucleotide. [0038] In other specific aspects, both the tagged oligonucleotide and the tag closing oligonucleotide are present in the nucleic acid sample during step (a), and wherein in step (a) the target hybridizing sequence favors hybridization to the target nucleic acid sequence rather than with tag closing oligonucleotide.

[0039] As highlighted above, any of a variety of amplification techniques can be used in the methods of the invention. In certain cases, it may be beneficial to use an isothermal amplification reaction, such as a transcription based amplification reaction, preferably TMA or TMA in real time.

[0040] In a particular aspect, the first oligonucleotide comprises an RNA polymerase promoter that is located 5 'to the hybridizing sequence. In another aspect, the second oligonucleotide comprises an RNA polymerase promoter that is located 5 'to the hybridizing sequence, and wherein the tagged oligonucleotide further includes an RNA polymerase promoter that is located 5' from the second region.

[0041] The target nucleic acid sequence amplified according to these methods may be any of the target nucleic acid sequences of interest, but will generally be in general a nucleic acid sequence obtained from the microorganism. Furthermore, the method can be selective for the amplification of the target nucleic acid sequence contained in the nucleic acid of a single strain or species of microorganisms or in many species of microorganisms. Alternatively, the method may be selective for the amplification of multiple target nucleic acid sequences contained in the nucleic acid of many species of microorganisms, wherein, for example, in step (a) the target hybridizing sequence from the tagged oligonucleotide hybridizes to the target region present in each of the numerous target nucleic acid sequences .

[0042] In another aspect, the method is selective for the amplification of a target nucleic acid sequence contained in each of a plurality of target nucleic acids and wherein in step (a) the target hybridizing sequence hybridizes to the 3'-end of the target nucleic acid sequence from each of the numerous acids nucleic acids present in the nucleic acid sample. In another aspect, the target nucleic acid sequence contained in each of these numerous target nucleic acids is the same nucleic acid sequence.

[0043] In a particular embodiment, the method is selective for the amplification of many bacterial or fungal target nucleic acid sequences, e.g., wherein said multiple bacterial or fungal target nucleic acid sequences are ribosomal nucleic acid sequences.

In a more particular embodiment, the method is selective for the amplification of target nucleic acid sequences obtained from organisms belonging to the group of bacterial species including Staphylococci species (e.g. Staphylococcus aureus, Staphylococcus epidermis and Staphylococcus haemalyticus), Steptococci species (e.g. Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci and beta-hemolytic streptococci), Enterococcus species (e.g. Enterococcus faeclum and Enterococcus faecalis), Escherichia species (e.g. Escherichia Klebi). Klebsiella oxytoca), Pseudomonas species (e.g. Pseudomonas aeruginosa), Enterobacter species (e.g. Enterobacter cloacae and Entero12 bacterial aerogenes), Proteus species (e.g. Proteus mirabilis), Bacterioides species, Clostridium species, Serratia species (e.g. Serratia marcescens), Acinetobacter species (e.g. Acinetobacter baumannii) and Stenotrophomonar species (e.g. Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0045] In another particular embodiment, the method is selective for the amplification of target nucleic acid sequences obtained from organisms belonging to the group of fungi species including Candida species (e.g. Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis and Candida famata), Histoplama capsulatum, Cryptococcus species (e.g. Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii) Coccidioides species (e.g. Coccidioides immitis), Trichosporon species (e.g. Trichosporon cutaneum), Malassezia species (e.g. Malassezia furfur), Rhodotorula species, Nocardia species, e.g. Nocardia asteria and Asperigillus species (e.g., Asperigillus fumigatus). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0046] In certain aspects of step (a), the target hybridizing sequence hybridizes to the 3'-end of each of the multiple target nucleic acid sequences present in the nucleic acid sample. Furthermore, in step (c) the first oligonucleotide hybridizes to the 3'-terminus of each of the many target nucleic acid sequences present in the nucleic acid sample.

[0047] The method may also comprise a plurality of first oligonucleotides, wherein in step (c) each of said plurality of first oligonucleotides hybridizes to the 3'end of the complement of at least one, but not all, of the many target nucleic acid sequences present in the nucleic acid sample .

[0048] In a particular embodiment of the invention, the tagged oligonucleotide is a universal bacterial oligonucleotide or a universal fungal oligonucleotide. Such labeled oligonucleotides are particularly well suited to sterility testing methods, such as methods for bioprocess material analysis or sepsis diagnostics.

[0049] In a more particular aspect, in step (a) the target hybridizing sequence hybridizes to the 3'-end of the target nucleic acid from each of the numerous target nucleic acids present in the nucleic acid sample, said multiple target nucleic acids belonging to the class of microorganisms selected from the group consisting of specific bacteria, Gram-positive bacteria, Gram-negative bacteria and fungi.

In another aspect, each of the numerous target nucleic acids is a ribosomal nucleic acid.

[0050] In another aspect of the disclosure, these multiple target nucleic acid sequences include sequences found in bacterial microorganisms from a class selected from the group consisting of Staphylococci species (e.g., Staphylococcus aureus, Staphylococcus epidermis and Staphylococcus haemolyticus), Steptococci species (e.g. Streptogenococci pneumonia) , Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci and beta-hemolytic streptococci), Enterococcus species (e.g. Enterococcus faecium and Enterococcus faecalis), Escherichia species (e.g. Escherichia coli), Klebsiella species (e.g. Klebsiella pneumonia and Klebsiella oxytoca), Pseudomonas species (e.g. Pseudomonas aeruginosa), Enterobacter species (e.g. Enterobacter cloacae and Enterobacter species Proteus (e.g. Proteus mirabilis), Bacterioides species, Clostridium species, Serratia species (e.g. Serratia marcescens), Acinetobacter species (e.g. Acinetobacter baumannii) and Stertotrophomonas species (e.g. Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0051] In another aspect of the disclosure, these numerous target nucleic acid sequences include those found in fungal microorganisms from a class selected from the group consisting of Candida species (e.g., Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides , Candida guilliermondi, Candida pseudotropicalis and Candida famata), Histoplama capsulatum, Cryptococcus species (e.g. Cryptococcus neoformans, Cryptococcus albidia and Cryptococcus laurentii) Coccidioides species (e.g. Coccidioides immitis), Trichosporon species (e.g. Trichosporon cutaneum), Malassezia species (e.g. Malassezia furfur), Rhodotorula species, Nocardia species, e.g. Nocardia asteria and Asperigillus species (e.g., Asperigillus fumigatus). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0052] Nucleic acid samples are often exposed to a known contaminant source of the target nucleic acid sequence after step (b) and the methods described accordingly ensure that the production of amplification products is essentially limited to the amplification of the target nucleic acid sequence provided in the nucleic acid sample rather than from sources of the contaminating target nucleic acid sequence. For example, one or more reagents or components used in the amplification reaction is a known contaminant source of the target nucleic acid sequence. Alternatively or additionally, one or more reagents are made of a material known to be a contaminating source of the target nucleic acid sequence, such as nucleic acid polymerases prepared using microorganisms known to contain the target nucleic acid sequence. In addition, the environmental conditions in which the method is performed may include a known source of contaminating the target nucleic acid sequence. In a particular aspect, at least a portion of this nucleic acid is obtained from a clinical, water, industrial, environmental, seed, beverage or food source.

[0053] According to another embodiment of the present disclosure, the target nucleic acid sequence is the target RNA sequence, and step (c) comprises: extending the tagged oligonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction using a DNA polymerase to produce a first primer extension product comprising a region complementary to the target nucleic acid sequence; separating the first primer extension product from the target nucleic acid sequence using an enzyme that selectively degrades that portion of the target nucleic acid sequence that is hybridized to the first primer extension product; treating the first primer extension product with the first oligonucleotide, wherein the first oligonucleotide is a promoter oligonucleotide comprising the first and second regions, wherein the first region comprises a hybridizing sequence that hybridizes with a region from the first primer extension product complementary to the 5'-end of the target nucleic acid sequence hybrid promoter oligonucleotide: the first primer extension product, and the second region comprises a promoter for RNA polymerase that is located 5 'to the first region; hybrid transcription from promoter oligonucleotide: the first primer extension product of multiple copies of the first RNA product complementary to at least a portion of the first primer extension product using RNA polymerase that recognizes the promoter and starts transcribing therefrom, the base sequence of the first RNA product being substantially identical to the sequence the base of the target nucleic acid sequence and the complement of the tag sequence; treating the first RNA product with a second oligonucleotide, wherein the second oligonucleotide is a priming oligonucleotide that hybridizes to the complement of the tag sequence to form a hybrid priming oligonucleotide: first RNA product, such that the primer extension reaction can be started from the priming oligonucleotide; primer oligonucleotide extension in a primer extension reaction using DNA polymerase to form a second primer extension product complementary to the first RNA product, wherein the second primer extension product has a 3'-end that is complementary to

The 5'-end of the first RNA product; separating the second primer extension product from the first RNA product using an enzyme that selectively degrades this first RNA product; treating the second primer extension product with a promoter oligonucleotide to form a hybrid promoter oligonucleotide: second primer extension product; extending the 3'-end of the second primer extension product in the promoter oligonucleotide hybrid: second primer extension product to attach a complementary sequence to the second promoter oligonucleotide region; and hybrid transcription from the promoter oligonucleotide: the second primer extension product of multiple copies of the second RNA product complementary to the second primer extension product using RNA polymerase, wherein the base sequence of the second RNA product is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the tag sequence.

[0054] In another aspect of this embodiment of the disclosure, step (a) further comprises treating the nucleic acid sample with a binding molecule that binds to the target nucleic acid adjacent to or near the 5'-end of the target nucleic acid sequence, and wherein the first extension product the primer has a 3'-end which is determined by the binding molecule and which is complementary to the 5'-end of the target nucleic acid sequence.

[0055] In another aspect, step (c) of the above embodiment further comprises extending the 3 'end of the first primer extension product in the hybrid promoter oligonucleotide: first primer extension product to attach a sequence complementary to the promoter. In yet another aspect, the promoter oligonucleotide is modified to prevent DNA synthesis from starting.

[0056] The promoter oligonucleotide hybridized to the first primer extension product is extended using DNA polymerase to produce a primer extension product complementary to the first primer extension product; and the promoter oligonucleotide hybridized to this second primer extension product is extended using DNA polymerase to produce a primer extension product complementary to the second primer extension product.

[0057] Separation steps in the methods described can be accomplished using ribonuclease activity provided by DNA polymerase. Alternatively, the separation steps are carried out using ribonuclease activity provided by an enzyme other than this DNA polymerase.

[0058] According to another embodiment of the present disclosure, the target nucleic acid sequence is the target RNA sequence, and step (c) comprises: elongation of the labeled olon16 gonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction using DNA polymerase to form a first primer extension product containing a region complementary to the target nucleic acid sequence, wherein the tagged oligonucleotide further comprises a third region located 5 'to the second region, which third region comprises a promoter for RNA polymerase; separating the first primer extension product from the target nucleic acid sequence using an enzyme that selectively degrades the portion of the target nucleic acid that is hybridized to the first primer extension product; treating the first primer extension product with the first oligonucleotide, wherein the first oligonucleotide is a priming oligonucleotide that hybridizes to the region of the first primer extension product complementary to the 5'-end of the target nucleic acid sequence to form the hybrid primer oligonucleotide: the first primer extension product, such that the extension reaction the primer may be started with a priming oligonucleotide; primer oligonucleotide extension in a primer extension reaction using DNA polymerase to form a second primer extension product complementary to the first primer extension product; and the use of a second primer extension product as a template for transcription of multiple copies of the first RNA product complementary to at least a portion of the second primer extension product using RNA polymerase which recognizes the promoter and initiates transcription therefrom, wherein the base sequence of the first RNA product is substantially identical to the sequence tag sequence bases and the complement of the target nucleic acid sequence.

[0059] In another aspect of this embodiment, step (c) further comprises: treating the first RNA product with a priming oligonucleotide to form a hybrid primer oligonucleotide: first RNA product, such that the primer extension reaction can be started from the priming oligonucleotide; primer oligonucleotide extension in a primer extension reaction using DNA polymerase to form a third primer extension product complementary to the first RNA product, which third primer extension product has a 3'-complement complementary to the 5'-end of the first RNA product; separating the third primer extension product from the first RNA product using an enzyme that selectively degrades the first RNA product; treating the third primer extension product with a second oligonucleotide, which second oligonucleotide is a promoter oligonucleotide comprising the first and second regions, wherein the first region comprises a hybridizing sequence that hybridizes to the complement of the tag sequence to form a hybrid promoter oligonucleotide: third primer extension product, such that the extension reaction the primer can be started from a promoter oligonucleotide, and wherein the second region comprises a promoter for RNA polymerase that is located 5 'to the first region; extending the promoter oligonucleotide in a primer extension reaction using DNA polymerase to form a fourth primer extension product complementary to the third primer extension product; extending the third primer extension product to add a sequence complementary to the promoter; hybrid transcription promoter oligonucleotide: a third primer extension product of multiple copies of a second RNA product complementary to a third primer extension product using an RNA polymerase that recognizes the promoter and begins transcription therefrom, the base sequence of the second RNA product being substantially identical to the base sequence of the tag sequence and completing the target nucleic acid sequence.

[0060] In another aspect of this embodiment, the separation steps are carried out utilizing the ribonuclease activity provided by the DNA polymerase. Alternatively, the separation steps are carried out using ribonuclease activity provided by an enzyme other than this DNA polymerase.

[0061] According to another embodiment of the present disclosure, the target nucleic acid sequence is the target DNA sequence, and step (c) comprises: extending the tagged oligonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction using a DNA polymerase to produce a first primer extension product comprising a region complementary to the target nucleic acid sequence; treating the first primer extension product with a first oligonucleotide, which first oligonucleotide is a promoter oligonucleotide comprising a first and second region, wherein the first region comprises a hybridizing sequence that hybridizes to the first primer extension product region that is complementary to the 5'-end of the target acid sequence nucleic acid to form a hybrid promoter oligonucleotide: the first primer extension product, and wherein the second region is a promoter for RNA polymerase that is located 5 'to the first region; hybrid transcription from promoter oligonucleotide: the first primer extension product of multiple copies of the first RNA product complementary to at least a portion of the first primer extension product using RNA polymerase that recognizes the promoter and starts transcribing therefrom, the base sequence of the first RNA product being substantially identical to the sequence the base of the target nucleic acid sequence and the complement of the tag sequence; treating the first RNA product with a second oligonucleotide, which second oligonucleotide is a primer oligonucleotide that hybridizes to the complement of the tag sequence to form a hybrid oligonucleotide primer: first RNA product, such that the primer extension reaction can be started from the priming oligonucleotide; primer oligonucleotide extension in a primer extension reaction using DNA polymerase to obtain a second primer extension product comprising a complement to the first RNA product, which second primer extension product has a 3'-end that is complementary to the 5'-end of the first RNA product; separating the second primer extension product from the first RNA product using an enzyme that selectively degrades the first RNA product; treating the second primer extension product with a promoter oligonucleotide to form a hybrid promoter oligonucleotide: second primer extension product; extending the 3'-end of the second primer extension product in the hybrid promoter oligonucleotide: second primer extension product to attach a promoter complementary sequence; and hybrid transcription from the promoter oligonucleotide: the second primer extension product of multiple copies of the second RNA product complementary to the second primer extension product using RNA polymerase, wherein the base sequence of the second RNA product is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the tag sequence.

[0062] In one aspect of this embodiment, the promoter oligonucleotide is modified to prevent DNA synthesis from starting.

[0063] In another aspect, step (a) further comprises: treating the displacement oligonucleotide with the target nucleic acid sample upstream of the tagged oligonucleotide such that the primer extension reaction can be started from the displacement oligonucleotide; and extending the displacer oligonucleotide in the primer extension reaction using DNA polymerase to form a third primer extension product that displaces the first primer extension product from the target nucleic acid.

[0064] In yet another embodiment, step (a) further comprises treating the nucleic acid sample with a binding molecule that binds to the target nucleic acid adjacent to or near the 5'-end of the target nucleic acid sequences, wherein the first primer extension product has The 3'-end which is determined by this binding molecule and which is complementary to the 5'-end of the target nucleic acid sequence.

[0065] In a more particular aspect, step (c) further comprises extending the 3'-end of the first primer extension product in the hybrid promoter oligonucleotide: first primer extension product to attach a sequence complementary to the promoter sequence.

[0066] In another particular aspect, step (c) further comprises: extending the promoter oligonucleotide hybridized to the first primer extension product using DNA polymerase to produce a starter extension product complementary to the first primer extension product; and extending the promoter oligonucleotide hybridized to the second primer extension product using DNA polymerase to produce a primer extension product complementary to the second primer extension product.

[0067] In one embodiment, the separation steps are carried out using the ribonuclease activity provided by this DNA polymerase. Alternatively, the separation steps are carried out using ribonuclease activity provided by an enzyme other than this DNA polymerase. [0068] Another embodiment of the present disclosure provides a kit for use in the selective amplification of at least one target nucleic acid sequence from a nucleic acid sample, which kit comprises: a labeled oligonucleotide comprising a first region comprising a target hybridizing sequence that hybridizes to the 3'-end of the target nucleic acid sequence in a first set of conditions, such that the first region can be extended in a matrix dependent manner in the presence of DNA polymerase, and a second region comprising the tag sequence located 5 'to the first region, which second region does not hybridize to the target nucleic acid comprising the target nucleic acid sequence in the first set of conditions; a tag closing sequence that hybridizes to a target hybridizing sequence in a second set of conditions, thereby blocking hybridization of the tagged oligonucleotide with the target nucleic acid sequence, which tag closing sequence does not hybridize stably with the sequence goal in the first set of conditions; and a first priming oligonucleotide that hybridizes to the complement of the tag sequence under a second set of conditions, such that the first priming oligonucleotide can be extended in a matrix dependent manner in the presence of DNA polymerase.

[0069] In a more particular aspect of this embodiment, the tagged oligonucleotide further comprises a third region comprising an RNA polymerase promoter, which third region is located 5 'to the second region.

[0070] In another aspect, the 3'-terminal base of the target hybridizing sequence hybridizes to the 5'-base of the tag closing sequence when the target hybridizing sequence is not hybridized to the target nucleic acid sequence under the second set of conditions.

[0071] In yet another aspect, the 5'-end of the tag closing sequence comprises a moiety for stabilizing the duplex formed between the tag closing sequence and the target hybridizing sequence when the target hybridizing sequence is not hybridized to the target nucleic acid sequence under the second set of conditions.

[0072] In another aspect, the tagged oligonucleotide and tag closing sequence are separate molecules, wherein the tag closing sequence is a tag closing oligonucleotide. Alternatively, the tagged oligonucleotide and tag closing sequence are contained in the same molecule.

[0073] The tag closing sequence may be linked to the tagged oligonucleotide via a non-nucleotide linker, for example a non-nucleotide linker containing at least one baseless nucleotide and polyethylene glycol.

[0074] In another aspect, the 3'-end of the tag closing sequence is joined to the 5'-end of the tagged oligonucleotide. Alternatively, the 5'-end of the tag closing sequence is joined to the 5'-end of the tagged oligonucleotide.

[0075] In yet another aspect, the tag closing sequence hybridizes to the target hybridizing sequence to form an anti-parallel duplex when the target hybridizing sequence is not hybridized to the target nucleic acid sequence under the second set of conditions.

[0076] In another aspect, the tag closing sequence is modified to prevent the initiation of DNA synthesis therefrom, for example by including a blocking moiety situated at its 3'-terminus.

[0077] In another aspect, the tag closing sequence hybridizes to the target hybridizing sequence to form a parallel duplex when the target hybridizing sequence is not hybridized to the target nucleic acid sequence under the second set of conditions. [0078] In yet another aspect, the duplex comprises a 3'-terminal base hybridizing sequence to a target hybridized to a 3'-terminal base of the tag closing sequence. [0079] The tag closing sequence, in this aspect, can be modified to prevent the initiation of DNA synthesis from it, for example by including a blocking moiety situated at its 3'-terminus.

[0080] In another aspect of this embodiment, the first priming oligonucleotide stably hybridizes to the target nucleic acid and thus participates in detectable amplification of the target nucleic acid sequence under a second set of conditions.

[0081] In another aspect, the kit further comprises a second priming oligonucleotide that hybridizes to the 5'-complement of the target nucleic acid sequence under the second set of conditions, such that the second priming oligonucleotide can be elongated in a matrix dependent manner in the presence of DNA polymerase.

[0082] In yet another aspect, the kit further comprises a promoter oligonucleotide comprising the first and second regions, wherein the first region comprises a hybridizing sequence that hybridizes to the 5'-complement of the target nucleic acid sequence under the second set of conditions, and the second region comprises a polymerase promoter RNA that is located 5 'from the first region.

[0083] In this aspect, the promoter oligonucleotide may be modified to prevent DNA synthesis from starting, for example, by incorporating a blocking moiety situated at its 3'-terminus.

[0084] In yet another aspect, the promoter oligonucleotide may be extended in a matrix-dependent manner in the presence of DNA polymerase when the hybridizing sequence is hybridized to the 5 'end of the target nucleic acid sequence under the second set of conditions.

[0085] The kits may also further comprise one or more reagents or components selected from one or more agents from DNA polymerase (such as reverse transcriptase), RNA polymerase, nucleoside triphosphates, a solid support substrate for binding a complex consisting of a target nucleic acid and labeled oligonucleotide. In another aspect, the tagged oligonucleotide is in free form in solution.

[0086] In another aspect, the kit does not include a restriction enzyme capable of cleaving the duplex formed between the tag closing sequence and the target hybridizing sequence under the second set of conditions.

[0087] In yet another aspect, the target hybridizing sequence hybridizes to the 3'-end of multiple target nucleic acid sequences under the first set of conditions.

[0088] In another embodiment, the tagged oligonucleotide is a universal bacterial oligonucleotide or a universal fungal oligonucleotide. For example, in one aspect, a target hybridizing sequence hybridizes to a target region at the 3'end of one or more target nucleic acid sequences, which target region is present in a number of microorganisms belonging to the class of microorganisms selected from the group consisting of specific bacteria, Gram-positive bacteria, Gram-negative bacteria and fungi in the first set of conditions. In another embodiment, the one or more target nucleic acid sequences are ribosomal nucleic acid sequences.

[0089] In a more specific aspect, the microorganisms belong to the class of bacterial microorganisms selected from the group consisting of Staphylococci species (e.g. Staphylo22 coccus aureus, Staphylococcus epidermis and Staphylococcus haemolyticus), Steptococci species (e.g. Streptococcus pneumoniae, Streptocococcus mycoccus) Viridons streptococci and beta-hemolytic streptococci), Enterococcus species (e.g. Enterococcus faecium and Enterococcus faecalis), Escherichia species (e.g. Escherichia coli), Klebsiella species (e.g. Klebsiella pneumoniae and Klebsiella oxytoca), Pseudomonas species (e.g. Pseudomonas aeruginosa), Enterobacter species (e.g. Enterobacier cloacaes) and Enterobacter Proteus (e.g. Proteus mirabilis), Bacterioides species, Clostridium species, Serratia species (e.g. Serratia marcescens), Acinetobacter species (e.g. Acinetobacter baumannii) and Stenotrophomonas species (e.g. Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0090] In another particular aspect, the microorganisms belong to a fungal group of microorganisms selected from the group consisting of Candida species (e.g., Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zey / anoides, Candida guilliermondi, Candida pseudotropical and Candida famata), Histoplama capsulatum, Cryptococcus species (e.g. Cryptococcus neoformans, Cryptococcus albidus and Cryptococcus laurentii), Coccidioides species (e.g. Coccidioides immitis), Trichosporon species (e.g. Trichosporon cutaneum), Malassezia species (e.g. Malassezia furfur), Rhodotorula species, Nocardia species (e.g. Nocardia asteroides), Fusarium species and Asperigillus species (e.g. Asperigillus fumigatus). At least some of these microorganisms would be suitable for detection in sepsis testing.

[0091] According to another embodiment of the disclosure, a reaction mixture is provided for amplifying a target nucleic acid sequence, which reaction mixture comprises: a tagged oligonucleotide comprising a first and a second region, wherein the first region comprises a target hybridizing sequence hybridized to the 3'-end of the target sequence nucleic acid, and the second region comprises a tag sequence located 5 'to the target hybridizing sequence; a first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-terminus of the complement of the target nucleic acid sequence; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of the tag sequence, wherein the unhybridized tagged oligonucleotide in the reaction mixture is in an inactive form that blocks or prevents hybridization of the unhybridized tagged oligonucleotide with the target nucleic acid sequence.

[0092] In a more specific aspect, according to this embodiment, the inactive form of the tagged oligonucleotide comprises a tag closing sequence hybridized to a target hybridizing sequence.

[0093] In another aspect, the tagged oligonucleotide and said tag closing sequence are separate molecules, wherein the tag closing sequence is the tag closing oligonucleotide.

[0094] In another aspect, the tagged oligonucleotide and tag closing sequence are contained in the same molecule.

[0095] In yet another aspect, the tagged oligonucleotide is not attached to a solid support.

[0096] Other specific embodiments of the disclosure relate to the use of the methods described herein as methods for monitoring bioprocess samples and streams, and the like. In one embodiment, for example, there is provided a method of monitoring a bioprocess for the presence of a contaminating nucleic acid, comprising the steps of: (a) acting on a first bioprocess sample with a tagged oligonucleotide, said tagged oligonucleotide comprising a first and a second region, wherein the first region comprises a target hybridizing sequence capable of hybridizing to a target nucleic acid sequence from the organism of interest and the second region comprises a tag sequence located 5 'to that of the target hybridizing sequence that does not stably hybridize to the target nucleic acid sequence; under conditions where the tagged oligonucleotide stably hybridizes to the target nucleic acid sequence present in this first sample; (b) removing or inactivating the unhybridized tagged oligonucleotide from the first bioprocess sample; and (c) exposing a second bioprocess sample, which second bioprocess sample includes the first bioprocess sample and further comprises additional bioprocess samples, to reagents and amplification conditions sufficient to amplify the target nucleic acid sequence using: (i) a first oligonucleotide that hybridizes to the complement of the tag sequence; and (ii) a second oligonucleotide that hybridizes to the complement of the target nucleic acid sequence, wherein the detectable amplification resulting from the first and second oligonucleotide occurs using the target nucleic acid sequence from the organism of interest first bioprocess sample, and not the target nucleic acid sequence provided in additional bioprocess samples.

[0097] In another embodiment, the present disclosure provides a method of monitoring a biological process for the presence of a contaminating nucleic acid, comprising the steps of: (a) treatment of a first bioprocess sample with a first tagged oligonucleotide, which first tagged oligonucleotide comprises a first and a second region, wherein the first region comprises a target hybridizing sequence capable of hybridizing to a target nucleic acid sequence from the organism of interest, and the second region comprises a first sequence label located 5 'to the target hybridizing sequence, which does not stably hybridize to the target nucleic acid sequence; under conditions where the first tagged oligonucleotide stably hybridizes to the target nucleic acid sequence present in this first sample; (b) treating the second bioprocess sample with a second tagged oligonucleotide, the second tagged oligonucleotide comprising the first and second regions, the first region comprising a target hybridizing sequence capable of hybridizing to a target nucleic acid sequence from the organism of interest, and the second region comprising the second sequence a tag located 5 'to the target hybridizing sequence and different from the first tag sequence, which does not stably hybridize to the target nucleic acid sequence; under conditions where the second tagged oligonucleotide stably hybridizes to the target nucleic acid sequence present in the second sample; and (c) performing a nucleic acid amplification reaction using a third bioprocess sample, which third bioprocess sample comprises a first and second bioprocess sample, using: (i) a first oligonucleotide that hybridizes to the complement of the first tag sequence; (ii) a second oligonucleotide sequence that hybridizes to the complement of the second tag sequence; and (iii) a third oligonucleotide that hybridizes to the complement of the target nucleic acid sequence, wherein detection of the amplification product derived from the first and second oligonucleotides is an indicator of the presence of the target nucleic acid sequence from the organism of interest in the first bioprocess sample, and wherein the detection of the amplification product derived from the first and third oligonucleotides is an indicator of the presence of the target nucleic acid sequence from the organism of interest in the second bioprocess sample.

[0098] In a further embodiment of the disclosure, a pre-amplification reaction mixture is provided for the selective amplification of one or more target nucleic acid sequences, which reaction mixture comprises: a tagged oligonucleotide comprising the first and second regions, said first region comprising a target hybridizing sequence hybridized to a target region contained at the 3'-end of one or more target nucleic acid sequences present in the reaction mixture, and the second region comprising a tag sequence located in the side 5 'from the target hybridizing sequence; a first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-terminus of the complement of one or more target nucleic acid sequences; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of the tag sequence, which second oligonucleotide preferably does not stably hybridize to the target nucleic acid containing the target nucleic acid sequence, so that it can be enzymatically extended in the presence of a nucleic acid polymerase added to the reaction mixture to form a primer extension product complementary to one or more target nucleic acid sequences, said reaction mixture being substantially free of the active form of the labeled oligonucleotide, which is not hybridized to a target region contained in one or more target nucleic acid sequences present in the reaction mixture, wherein the active form of the tagged oligonucleotide has an available hybridizing sequence with a target for hybridization with the target region present in the non-target nucleic acid added to the reaction mixture, and wherein the reaction mixture does not include a nucleic acid polymerase capable of extending any of the oligonucleotides in a matrix-dependent manner. The "non-target" nucleic acid is derived from a source outside the reaction mixture and may contain a sequence identical to that of the target nucleic acid sequence. The source of the non-target nucleic acid may be the environment, or it may be a component or reagent added to the reaction mixture, such as nucleic acid polymerase. The tagged oligonucleotide may be a tagged priming oligonucleotide or a tagged promoter oligonucleotide comprising a promoter recognized by an RNA polymerase located 5 'to the second region.

[0099] In one aspect of this embodiment, the tagged priming oligonucleotide does not include an RNA polymerase promoter, while the first oligonucleotide comprises an RNA promoter located 5 'to the hybridizing sequence of the first oligonucleotide. In a preferred aspect, the first oligonucleotide further comprises a blocking moiety situated at its 3'-end. In another aspect, which is useful for amplifying the target sequence from E. coli, the target hybridizing sequence from the tagged oligonucleotide consists of SEQ ID NO: 19, its corresponding RNA or their complement, and the first hybridizing sequence from the first oligonucleotide consists of SEQ ID NO: 20, its corresponding RNA or their complement.

[0100] In another aspect of this embodiment, the tagged oligonucleotide and first oligonucleotide may each separately comprise a RNA polymerase promoter located 5 'to the tag and hybridizing sequence, respectively. In this aspect, the first oligonucleotide may comprise a blocking moiety located at its 3'-terminus.

[0101] In yet another aspect of this embodiment, the tagged oligonucleotide comprises a tag closing sequence attached to its 3'-terminus, thereby forming a unitary molecule called "tag molecule". Depending on the nature of the amplification reaction, the tag molecule may or may not include the RNA polymerase promoter located 5 'to the tag sequence. In one embodiment, the tag molecules that have not hybridized to a target region from at least one target nucleic acid sequence remain "free" in the reaction mixture (ie, the tag molecules do not form hybrid duplexes other than by self-hybridization). Self-hybridized tag molecules are called "hairpin tag molecules," which is the inactive form of the tag molecule that prevents it from hybridizing with any complementary nucleic acids that are sequentially added to the reaction mixture, such as in the form of an impure enzyme preparation or a reagent containing non-target nucleic acids. In yet another aspect of this embodiment, substantially all of the tag molecules in the reaction mixture are in a hybridized state (hybridized either to the target region of the target nucleic acid sequence or to each other in the form of tag molecules with a hairpin structure). At least a portion of the tag molecules that have not hybridized to the target region of the target nucleic acid sequence (i.e. hairpin marker molecules) are removed from the reaction mixture, for example, by subjecting the reaction mixture to a directed capture and rinse procedure.

[0102] In yet another aspect of this embodiment, there is essentially no tagged oligonucleotides that exist in an unhybridized state when the reaction mixture is exposed to an enzyme preparation for the amplification of one or more nucleic acid sequences. Thus, in this aspect, the reaction mixture is substantially free of unhybridized tagged oligonucleotides specific for one or more target nucleic acid sequences provided in the sample of interest. This can be accomplished, for example, by a directed capture and wash procedure that separates hybridized tagged oligonucleotides from unhybridized tagged oligonucleotides, and then selectively removes unhybridized tagged oligonucleotides from the reaction mixture.

[0103] In yet another aspect of this embodiment, the tagged oligonucleotide does not comprise either a tag closing sequence or a tag closing oligonucleotide. Thus, in this aspect, the tagged oligonucleotide cannot be characterized as a "tag molecule."

[0104] In yet another aspect of this embodiment, a probe for detecting an amplification product synthesized in an in vitro reaction that includes enzymatic extension of the labeled oligonucleotide and second oligonucleotide is included. The amplification product contains copies of one or more nucleic acid sequences and / or their complement. [0105] In yet another embodiment, a reaction mixture is provided for selectively amplifying one or more target nucleic acid sequences, which reaction mixture comprises: a tagged oligonucleotide comprising the first and second regions, the first region comprising a target hybridizing sequence hybridized to the 3'-end of the target nucleic acid sequence, and the second region comprising a tag sequence situated 5 'to the target hybridizing sequence; a first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-terminus of the complement of the target nucleic acid sequence; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of the tag sequence, which second oligonucleotide preferably does not stably hybridize to the target nucleic acid containing the target nucleic acid sequence, so that it can be enzymatically extended in the presence of a nucleic acid polymerase added to or present in the reaction mixture to form a primer extension product complementary to one or more target nucleic acid sequences, and wherein substantially all of the unhybridized tagged oligonucleotide in the reaction mixture is in inactive form, which blocks or prevents hybridization of this unhybridized tagged oligonucleotide with the target nucleic acid sequence.

[0106] The inactive form of the tagged oligonucleotide may comprise a tag closing sequence hybridized with the target hybridizing sequence. The tag closing sequence may be a separate molecule when it is not hybridized to the target hybridizing sequence or it may be contained in a molecule comprising a tagged oligonucleotide, in which case the tag closing sequence is preferably connected to the 5'-end of the tagged oligonucleotide by non-nucleotide linker (i.e., the components of the linker cannot be amplified by nucleic acid polymerase). The tagged oligonucleotide may or may not be attached to a solid support and is preferably not directly attached to a solid support (e.g., particles or beads). If attached to a solid support, directly or indirectly, the tagged oligonucleotide may further function as an capture probe for binding and immobilizing the target nucleic acid sequence.

[0107] The labeled oligonucleotides from the above forms of reaction mixtures may have the characteristics of any of the above described forms of the various labeled oliginucleotides. And also, unless specifically excluded, the reaction mixtures may further include reagents and components needed to perform the amplification reaction.

[0108] These and other features and advantages of the present disclosure will become apparent upon reading the detailed description below, the accompanying drawings and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS [0109]

FIG. 1 illustrates the steps of a transcription-based amplification reaction initiated from a tagged priming oligonucleotide that hybridizes to the 3'-end of the target RNA sequence. The first extension product formed from the tagged priming oligonucleotide has a 3'-end that is determined by the terminating oligonucleotide hybridized adjacent to or near the 5'-end of the target RNA sequence. The blocked promoter oligonucleotide hybridizes to the 3'-end of the first extension product and is used to generate RNA transcripts that enter amplification cycles.

FIG. 2 illustrates the use of a hairpin tag molecule in the amplification reaction of FIG. 1.

Figures 3A and 3B illustrate the steps of a transcription-mediated amplification reaction that begins with a tagged promoter oligonucleotide that hybridizes to the 3'-end of the target RNA sequence.

FIG. 4 illustrates the use of a hairpin tag molecule in the amplification reaction of Figures 3A and 3B.

FIG. 5 illustrates the transcription-based amplification reaction steps initiated from a tagged priming oligonucleotide that hybridizes to the 3'-end of a single-stranded target DNA sequence. The first extension product formed from the tagged priming oligonucleotide has a 3'-end that is determined by the terminating oligonucleotide hybridized adjacent to or near the 5'-end of the target DNA sequence. The displacement oligonucleotide hybridized 5 'to the tagged priming oligonucleotide is extended to form a second extension product that displaces the first extension product from the target DNA sequence. The blocked promoter oligonucleotide hybridizes to the 3'-end of the first extension product and is used to generate RNA transcripts that enter amplification cycles.

FIG. 6 illustrates the use of a hairpin tag molecule in the amplification reaction of FIG. 5.

FIG. 7 illustrates the polymerase chain reaction steps that are initiated from a tagged priming oligonucleotide that hybridizes to the target DNA sequence.

FIG. 8 illustrates the use of a hairpin tag molecule in the amplification reaction of FIG. 7.

FIG. 9 illustrates reverse transcription polymerase chain reaction steps initiated from a tagged priming oligonucleotide that hybridizes to the target RNA sequence.

FIG. 10 illustrates the use of a hairpin tag molecule in the amplification reaction of FIG. 9.

FIG. 11 illustrates a single, 3'-blocked tag closing oligonucleotide hybridized in an anti-parallel manner to the 3'-end of a tagged priming oligonucleotide, thereby blocking hybridization of the tagged priming oligonucleotide to the target nucleic acid sequence.

FIG. 12 illustrates a single, 3'-blocked tag closing oligonucleotide hybridized in an anti-parallel manner to the 3'-end of a tagged promoter oligonucleotide, thereby blocking hybridization of the tagged promoter oligonucleotide with the target nucleic acid sequence.

FIG. 13 illustrates a hairpin tag molecule that includes a 3'-blocked tag closing sequence hybridized in parallel with the 3'-end of a tagged priming oligonucleotide, thereby blocking hybridization of the tagged priming oligonucleotide with the target nucleic acid sequence. The 5'-end of the tag closing sequence is joined to the 3'-end of the tagged priming oligonucleotide via a non-nucleotide linker.

FIG. 14 illustrates a hairpin tag molecule that includes a 3'-blocked tag closing sequence hybridized in parallel with the 3'-end of a tagged promoter oligonucleotide, thereby blocking the hybridization of the tagged promoter oligonucleotide with the target nucleic acid sequence. The 5'-end of the tag closing sequence is joined to the 3'-end of the promoter sequence from the tagged promoter oligonucleotide via a non-nucleotide linker.

FIG. 15 illustrates a hairpin tag molecule that includes a 3'-blocked tag closing sequence hybridized in an antiparallel manner to the 3'-end of a tagged priming oligonucleotide, thereby blocking hybridization of the tagged priming oligonucleotide to a target nucleic acid sequence. The 5'-end of the tag closing sequence is joined to the 3'-end of the tag sequence from the tagged priming oligonucleotide via a non-nucleotide linker.

FIG. 16 illustrates a hairpin tag molecule that includes a 3'-blocked tag closing sequence hybridized in an antiparallel manner to the 3'-end of a tagged promoter oligonucleotide, thereby blocking hybridization of the tagged promoter oligonucleotide with the target nucleic acid sequence. The 5'-end of the tag closing sequence is joined to the 3'-end of the promoter sequence from the tagged promoter oligonucleotide via a non-nucleotide linker.

FIG. 17 shows the raw curves for HCV amplifications in which no target was administered to the amplification reagent. Detectable amplification did not occur when the HCV transcript was not administered to the targeted uptake or amplification reagents, while the average time T for reactions containing 1 x 10⁶copy of the HCV transcript in the targeted uptake reagent was 6.3 minutes.

FIG. 18 shows the raw curves for HCV amplifications in which the target was administered to the amplification reagent. Detectable amplification did not occur when HCV transcript was given to the amplification reagent, while the average time T for reactions containing 1 x 10⁶ copy of the HCV transcript in the targeted uptake reagent was 6.3 minutes. Zero samples at directed uptake did not amplify, even in the presence of 1 million copies of HCV 1a administered to the amplification reagent.

FIG. 19 shows the raw curves for HCV amplifications in which the target and labeled non-T7 primer were administered to the amplification reagent. Avg. the T time for 1 million copies of the HCV 1a target present only in the targeted capture step with the labeled non-T7 primer and the terminating oligonucleotide administered to the amplification reagent was 7.2 minutes. Zero samples in targeted uptake with target, terminating oligonucleotide and labeled primer other than with T7 administered to the amplification reagent also resulted in strong amplification with an average time of T = 8.6 minutes.

FIG. 20 is a graph showing results from monitoring versus reaction time of a nucleic acid amplification that included 0 or 10⁶ copy of synthetic E. coli rRNA template. A thin dashed line shows the results for a reaction carried out using 0 copies of template, and a thick solid line shows the results for a reaction carried out using 10⁶ matrix copy.

FIG. 21 is a graph showing results from monitoring versus time of nucleic acid amplification reaction, which included 0, 10³ or 10⁵ copy of synthetic E. coli rRNA template.

DETAILED DESCRIPTION OF THE INVENTION [0110] Amplification methods are provided in accordance with the present invention that preferably limit or eliminate false positive amplification signals resulting from a contaminating biological material that may be present in an amplification reagent or component. The methods provided also allow for less stringent purification and sterilization procedures that were typically needed to ensure that enzymes and other reagents or components used in the amplification reactions, and the environment in which the amplification reactions are carried out, are free from contamination by microorganisms or their components, such as nucleic acid material, that can give false positive results.

[0111] In carrying out the present invention, unless otherwise indicated, standard techniques of molecular biology, recombinant DNA and chemistry will be used, all within the range of skill in the art. Such techniques are fully explained in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd Edition, Sambrook et al., Edited by Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (DN Glover ed., 1985); Oligonucleotide Synthesis (MJ Gait ed., 1984); Mullis et al., US Patent No. 4,683195; Nucleic Acid Hybridization (BD Hames and SJ Higgins ed. 1984);

B. Perbal, A Practical Guide To Molecular Cloning (1984); treatise, Methods In Enzymology (Academic Press, Inc., NY); and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989).

Definitions [0112] The following terms have the following meanings, unless explicitly stated otherwise. Note that if an object is listed in the singular, it refers to one or more of these objects; for example, the term "nucleic acid" is understood to mean one or more nucleic acids. Thus, singular listing of the object and the terms "one or more" and "at least one" may be used interchangeably herein.

Nucleic acid [0113] The term "nucleic acid" is intended to include the singular "nucleic acid" as well as the plural "nucleic acids", and refers to any chain of two or more nucleotides, nucleosides or nucleic bases (e.g. de32 oxy-ribonucleotides or ribonucleotides) covalently linked to each other. Nucleic acids include, but are not limited to, viral genomes, or parts thereof, of DNA or RNA, bacterial genomes or parts thereof, fungal, plant or animal genomes, or parts thereof, informational RNA (mRNA), ribosomal RNA (rRNA), transporting RNA (tRNA), plasmid DNA, mitochondrial DNA or synthetic DNA or RNA. The nucleic acid may be provided in linear (e.g., mRNA), circular (e.g., plasmid), or branched form, as well as in double-stranded and single-stranded form. Nucleic acids may contain modified bases to change the function or behavior of the nucleic acid, e.g., by attaching a 3'-terminal dideoxynucleotide to block additional nucleotide attachment to the nucleic acid. As used herein, the term "nucleic acid sequence" refers to the base sequence that forms the nucleic acid. The term "polynucleotide" can be used herein to designate a nucleic acid chain. Throughout this application, nucleic acids are designated herein having a 5'-end and 3'-end. Standard nucleic acids, e.g., DNA and RNA, are usually synthesized "3'-to-5", ie, by attaching nucleotides to the 5'-end of the elongating nucleic acid.

[0114] The term "nucleotide" means a nucleic acid subunit consisting of a phosphate group, a 5-carbon sugar and a nitrogen base. Ribose is a 5-carbon sugar found in RNA. In DNA, the 5-carbon sugar is 2'-deoxyribose. The term also includes analogues of such subunits, such as the methoxy group at the 2 'position of ribose (2'-O-Me). As used herein, methoxy-oligonucleotides containing "T" residues have a methoxy group at the 2 'position of the ribose moiety and uracil at the base position of the nucleotide.

[0115] The term "non-nucleotide unit" means a unit that does not significantly contribute to polymer hybridization. For example, such units must not participate in any significant hydrogen bond to the nucleotide and will not include units having as component one of five nucleotide bases or analogues thereof.

Target nucleic acid / target sequence [0116] The term "target nucleic acid" means the nucleic acid present in a nucleic acid sample, including the "target sequence" to be amplified. The target nucleic acids can be DNA or RNA as described herein, and they can be single-stranded or double-stranded. The target nucleic acid may include other sequences other than the target sequence, which may not be amplified. Typical target nucleic acids include viral genomes, bacterial genomes, fungal genomes, plant genomes, animal genomes, rRNA, tRNA or mRNA from viruses, bacteria or eukaryotic cells, mitochondrial DNA or chromosomal DNA.

[0117] Target nucleic acids can be isolated from any number of sources based on the purpose of the amplification assay being performed. Sources of target nucleic acids include, but are not limited to, clinical samples (e.g. blood, whole blood or platelets, urine, saliva, feces, semen or spinal fluid), environmental samples (e.g. water or soil samples), food and drink samples , industrial samples (e.g. process products and materials (including water), seed stocks, cDNA libraries or whole cellular RNA. By "isolated" is meant that the sample containing the target nucleic acid is taken from its natural environment; however, this term is not associated with any particular degree of purification. When necessary, the target nucleic acids of the present invention are made available for interaction with various oligonucleotides of the present invention. This may include, for example, cell lysis or cell permeabilization to release the target nucleic acid from the cells, after which one or more purification steps may be performed, such as a series of isolation and washing steps. See, e.g., Clark et al., "Method for Extracting Nucleic Acids from a Wide Range of Organisms," US Patent No. 5,786,208; and Hogan, "Polynucleotide Matrix-Based Method of Identifying Microorganisms," US Patent No. 6821770. This may be particularly important when the sample source or cellular material released into the sample may interfere with the amplification reaction. Methods for preparing target nucleic acids from various sources for amplification are well known to those of ordinary skill in the art. Target nucleic acids of the present invention may be purified to some extent before the amplification reactions described herein, but in other cases, the sample is added to the amplification reaction without any further manipulation.

[0118] The term "target sequence" refers to a particular nucleotide sequence from a target nucleic acid to be amplified. The term "target sequence" includes complexing sequences with which oligonucleotides (e.g., tagged oligonucleotides, primer oligonucleotides and / or promoter oligonucleotides) enter into complexes during the processes of the present invention. When the target nucleic acid is originally single stranded, the term "target sequence" will also refer to a sequence complementary to the "target sequence" as present in the target nucleic acid. When the "target nucleic acid" is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. When choosing a target sequence, one of skill in the art will understand that a "unique" sequence should be selected to distinguish between unrelated and closely related target nucleic acids. As will be understood by those of ordinary skill in the art, "unique" sequences are evaluated in relation to the test environment. At least those sequences that are recognized by the target hybridizing sequence from the tagged oligonucleotide and the associated probe or detection probes (as described in more detail elsewhere herein) should be unique in the environment under study, but need not be unique within the pool of all possible sequences . Furthermore, although the target sequence should contain a "unique" sequence for recognition by a tagged oligonucleotide or detection probe, not always the priming oligonucleotide and / or promoter oligonucleotide recognize "unique" sequences. In some embodiments, it may be desirable to select a target sequence that is common to a class of organisms, for example, a sequence that is common to all strains

E. coli, which may be in the sample. In other situations, a very high specific target sequence or target sequence having at least a highly specific region recognized by the detection probe will be selected to distinguish between closely related organisms, for example pathogenic and non-pathogenic E. coli. The target sequence of the present invention may be of any practical length. The minimum target sequence includes the region that hybridizes to the target hybridizing sequence from the tagged oligonucleotide, the complement of the region that hybridizes to the priming oligonucleotide or the promoter oligonucleotide hybridizing region, and the region used for detection, e.g., the region (or its complement) with the hybrid detection as described in more detail elsewhere herein. The region that hybridizes with the detection probe may coincide with or be contained within a region that hybridizes with the priming oligonucleotide (or its complement) or the region hybridizing with the promoter oligonucleotide (or its complement). In addition to the minimum requirements, the optimal length of the target sequence depends on several elements contemplated, for example, the number of secondary structures or self-hybridizing regions in the sequence. Determining the optimal length is easily achievable by those of ordinary skill in the art using routine optimization methods. Typically, the target sequences of the present invention range from 100 nucleotides in length to from about 150 to about 250 nucleotides in length. The optimal or preferred length may vary under different conditions, which can easily be tested by one of ordinary skill in the art in accordance with the methods described herein. The term "amplicon" refers to a nucleic acid molecule generated during an amplification procedure that is substantially complementary to or identical to the sequence contained within the target sequence. The term "amplification product" refers to an amplicon or some other product that is an indicator of the amplification reaction.

Oligonucleotides [0119] As used herein, the term "oligonucleotide" or "oligo" or "oligomer" is intended to include a single "oligonucleotide" as well as multiple "oligonucleotides", and refers to any polymer of two or more nucleotides , nucleosides, nucleic bases or related compounds, used as a reagent in the amplification methods of the present invention, as well as subsequent detection methods. Oligonucleotides may be from DNA and / or RNA and / or their analogs. The term oligonucleotide does not specify any particular function of the reagent, rather it is used in general so that it applies to all such reagents described herein. An oligonucleotide can perform many different functions, e.g. it can act as a primer if it is capable of hybridizing with a complementary strand and can be further extended in the presence of a nucleic acid polymerase, it can provide a promoter if it contains a sequence recognized by RNA polymerase and allows transcription, and may have the function of preventing hybridization or inhibiting primer extension if it is properly located and / or modified. Specific oligonucleotides of the present invention are described in more detail below. As used herein, an oligonucleotide can be of virtually any length, limited only by its specific activity in the amplification reaction or in the detection of the amplification product from the amplification reaction.

[0120] Oligonucleotides with a defined sequence and chemical structure can be produced by techniques known to those of ordinary skill in the art, such as chemical or biochemical synthesis and expression in vitro or in vivo from recombinant nucleic acid molecules, e.g., bacterial or viral vectors. As intended in this disclosure, the oligonucleotide does not consist solely of wild type chromosomal DNA or its transcription products in vivo.

[0121] Oligonucleotides may be modified in any manner, provided that the modification is compatible with the desired function of the given oligonucleotide. One of ordinary skill in the art can easily determine if a given modification is suitable or desirable for any given oligonucleotide of the present invention. Modifications include base modifications, sugar modifications, or backbone modifications. Base modifications include, but are not limited to, the use of the following bases in addition to adenine, cytosine, guanine, thymine and uracil: C-5 propyn, 2-aminoadenine, 5-methylcytidine, inosine, and dP and dK bases. Sugar groups from nucleoside subunits can be ribose, deoxyribose and analogs thereof, including, for example, ribonucleosides with 2'-O-methyl (2'-O-ME) substitution on the ribofuranose moiety. See Becker et al., "Method for Amplifying Target Nucleic Acids Using Modified Primers," US Patent No. 6,133,038. Other sugar modifications include, but are not limited to, 2'-amine, 2'-fluoro, (L) -alpha modifications treofuranosyl and pentopuranosyl. Nucleoside subunits can be linked by bonds, such as phosphodiester bonds, modified bonds or non-nucleotide moieties that do not prevent the oligonucleotide from hybridizing to its complementary target nucleic acid sequence. Modified bonds include those in which the standard phosphodiester linkage is replaced by another linkage, such as a phosphorothioate linkage or a methyl phosphate linkage. Nucleic base subunits can be connected, for example, by replacing the natural deoxyribosophosphate DNA main chain with a pseudo-peptide main chain, such as a 2-aminoethylglycine main chain, to which nucleic base subunits are attached a carboxymethyl linker with a central secondary amine. (DNA aalogs with a pseudo-peptide backbone are commonly called "peptide nucleic acids" or "PNA" and are disclosed by Nielsen et al., "Peptide Nucleic Acids", US Patent No. 5,5390,82.). Other binding modifications include, but are not limited to, morpholine bonds.

[0122] Non-limiting examples of oligonucleotides or oligomers contemplated by the present invention include nucleic acid analogues containing bicyclic and tricyclic nucleoside and nucleotide (LNA) analogs. See Imanishi et al., "Bicyclonucleoside and Oligonucleotide Analogues", US Patent No. 6,268490; and Wengel et al., "Oligonucleotide Analogues," US Patent No. 6,770,461.) Any nucleic acid analog is contemplated in the present invention, provided that the modified oligonucleotide can perform its intended function, e.g. hybridize to the target nucleic acid under stringent hybridization or amplification conditions, or interact with DNA or RNA polymerase, thereby initiating extension or transcription. In the case of detection probes, modified oligonucleotides must also be capable of preferentially hybridizing with the target nucleic acid under stringent hybridization conditions.

[0123] While the structure and sequence of oligonucleotides for the present invention depend on their function, as described below, several variables need to be considered in general. Among the most important are length, melting point (Tm), specificity, complementarity to other oligonucleotides in the system, G / C content, polypyrimidine (T, C) or polypurine (A, G) and 3'-end sequence. Control of these and other variables is a standard and well-known aspect of oligonucleotide design, and various computer programs are readily available to screen large numbers of potential oligonucleotides for optimal ones.

[0124] The 3 'end of the oligonucleotide (or other nucleic acid) may be blocked in a variety of ways using a blocking moiety as described below. The "blocked" oligonucleotide is not effectively extended by attaching the nucleotides to its 3'-end by DNA or RNA-dependent DNA polymerase to form a complementary DNA strand. As such, a "blocked" oligonucleotide cannot be a "primer". [0125] As used in the disclosure, the expression "an oligonucleotide having a nucleic acid sequence" comprising "," consisting of ", or" consisting essentially of "a sequence selected from" a group of specific sequences means that the basic and new feature An oligonucleotide is the ability to stably hybridize with a nucleic acid having the exact complement of one of the listed nucleic acid sequences from the group under stringent hybridization conditions. The exact complement includes the appropriate DNA or RNA sequence.

[0126] The expression "oligonucleotide substantially corresponding to the nucleic acid sequence" means that said oligonucleotide is sufficiently similar to the reference nucleic acid sequence, such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence in that it will hybridize to the same target nucleic acid sequence under stringent hybridization conditions.

[0127] It will be understood by one of ordinary skill in the art that "substantially corresponding" oligonucleotides of the invention may differ from the reference sequence and still hybridize to the same target nucleic acid sequence. This difference from a nucleic acid can be expressed as the percentage of identical bases in the sequence or the percentage of perfectly complementary bases between the probe or primer and their target sequence. Thus, the oligonucleotide of the present invention generally corresponds to a reference nucleic acid sequence if these percentages of base identity or complementarity are from 100% to about 80%. In preferred embodiments, the percentage is from 100% to about 90%; in other preferred embodiments, the percentage is from 100% to about 95%. One skilled in the art will be aware of various modifications of hybridization conditions that may be required at different percentages of complementarity to allow hybridization to a specific target sequence without causing an unacceptable level of nonspecific hybridization.

Tagged oligonucleotide / heterologous tag sequence [0128] The term "tagged oligonucleotide" as used herein refers to an oligonucleotide that includes at least a first region and a second region, wherein the first region includes a "target hybridizing sequence" that hybridizes with 3 'end of the target nucleic acid sequence of interest, and wherein the second region comprises a "tag sequence" located 5 'to the target hybridizing sequence and which does not stably hybridize or bind to a target nucleic acid comprising the target nucleic acid sequence. The hybridization of the target hybridizing sequence to the target nucleic acid sequence results in a "tagged target nucleic acid sequence". The features and considerations regarding the construction of the target hybridizing sequence component will be the same as discussed above for the priming oligonucleotides.

[0129] The term "tag sequence" or "heterologous tag sequence" can mean essentially any sequence as long as it does not stably hybridize to the target nucleic acid sequence of interest and thus does not participate in detectable amplification. The tag sequence preferably does not stably hybridize with any sequence derived from the genome of the test organism or more particularly with any target nucleic acid under the reaction conditions. The tag sequence that is present in the tagged oligonucleotide is preferably designed so that it does not substantially impair or interfere with the ability of the hybridizing sequence to hybridize to its target sequence. In addition, the tag sequence will have sufficient length and composition such that, after embedding the tag sequence complement to the initial DNA extension primer product, the tag specific primer oligonucleotide can then be used to participate in subsequent amplification rounds as described herein. The tag sequence of the present invention is usually at least 10 nucleotides in length and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Those skilled in the art will recognize that tag sequence and tagged oligonucleotide design can be carried out according to any of a variety of convenient strategies while still achieving the goals and benefits described herein.

[0130] In certain embodiments, the tagged oligonucleotide is a "tagged priming oligonucleotide" comprising a tag sequence and a target hybridizing sequence. In other embodiments, the tagged oligonucleotide is a "tagged promoter oligonucleotide" comprising a tag sequence, a target hybridizing sequence, and a promoter sequence located 5 'to the tag sequence and effective to initiate transcription therefrom.

Inactivation [0131] The term "inactivation" means that the heterologous tag sequence is changed in such a way that it does not stably bind to the target nucleic acid sequence under amplification conditions. In the case of an unhybridized tagged oligonucleotide, the term "inactivation" means that the tagged oligonucleotide changes from an "active" conformation that allows the target hybridizing sequence to hybridize with the target nucleic acid sequence into an "inactive" conformation that blocks or otherwise prevents sequence hybridization hybridizing to the target with the target nucleic acid sequence. For example, the inactive conformation may be formed under such stringency conditions that allow the tag closing sequence to form a stable hybrid with the target hybridizing sequence (e.g., under less stringent conditions than the conditions for forming the active conformation of the tagged oligonucleotide). If it is not further changed, the hybrid tag closing sequence: the target hybridizing sequence remains closed under amplification conditions. Alternatively, the duplex formed between the tag closing sequence and the target hybridizing sequence may be altered by an enzyme such as DNAse, S1 nuclease, endonuclease such as a restriction enzyme which cleaves the double-stranded restriction site formed between the tag closing sequence and the target hybridizing sequence, ribonuclease activity (e.g. RNAse H activity) to digest the RNA component (e.g. target hybridizing sequence) from a DNA: RNA hybrid or exonuclease with 3'-to-5 'or 5'-to-3' activity to remove nucleotides from the target hybridizing sequence hybridized with the tag closing sequence. However, to avoid exposure of the sample to a potentially contaminating source of the target nucleic acid sequence, the use of enzymes to inactivate tagged oligonucleotides that have not hybridized to the target nucleic acid sequence is usually not preferred. Other methods of inactivation include chemical compounds for altering the target hybridizing sequence in such a way that it is unable to hybridize to the target nucleic acid sequence under amplification conditions.

[0132] Certain moieties may be included in the tag hybridization sequence to further stabilize hybrids formed between the target closing sequence and the target hybridizing sequence from tagged oligonucleotides, especially when at least some of the inactive tagged oligonucleotides are expected to be introduced into the amplification reaction mixture . Such convenient moieties include modified nucleotides, including LNA, 2'-O-ME-ribonucleotides, 2.6 diaminopurine, 5-methylcytosine and C-5 propynylcytosine or uracil. Those skilled in the art will be able to quickly select the number and position of such modified nucleotides to limit breathing at the 5 'and 3' ends of the tag closing sequence and to achieve the desired hybrid melting temperature without performing additional experiments. Other convenient moieties that engend with small furrow and outliers such as purine, DABCYL, pyridine and a 5'-trimethoxystylene cap.

Removal [0133] As used herein, the term "removal" refers to the physical separation of tagged target nucleic acid sequences from unhybridized tagged oligonucleotides. Tagged target nucleic acid sequences can be physically separated from unhybridized tagged oligonucleotides (or heterologous tag sequences) present in the nucleic acid sample by various techniques known to those of skill in the art. As an example, the labeled target nucleic acid sequence can be bound to a solid support and immobilized in the nucleic acid sample while unbound material is removed. To remove unbound material, the solid support may be subjected to one or more rinse / wash steps. Wash steps are intended to remove remaining unhybridized tagged oligonucleotides and potentially interfering cellular material or sample material. The washing step is usually added when the washing solution contains a component that inhibits amplification when present in a sufficiently high concentration, such as a detergent. The solid support preferably binds specifically to target nucleic acids or tagged target nucleic acid sequences to prevent unhybridized tagged oligonucleotide (or unbound heterologous tag sequences to enter the amplification reaction. Exemplary methods of capturing, immobilizing and purifying target nucleic acids are discussed below, an example of which is disclosed by Weisburg et al., "Two-Step Hybridization and Capture of a Polynucleotide", US Patent No. 6,534,273.

Tag closing sequence / tag closing oligonucleotide [0134] The terms "tag closing sequence" and "tag closing oligonucleotide" refer to an oligonucleotide that is complementary to a fragment of a target hybridizing sequence from a tagged oligonucleotide. The length and sequence of the tag closing sequence are selected such that the tag closing sequence does not stably hybridize with the target hybridizing sequence from the tagged oligonucleotide in the first set of conditions allowing stable hybridization of the target hybridizing sequence with the target sequence. The tag closing sequence may include baseless or base mismatch nucleotides with a target hybridizing sequence. If the tagged oligonucleotide is not hybridized to the target sequence, the tag closing sequence stably hybridizes to the target hybridizing sequence in a second set of milder conditions, thereby "inactivating" the tagged oligonucleotide or blocking its hybridization with the target sequence. The tag closing sequence may be in the form of a separate oligonucleotide or it may be joined to the 5'-end of the tagged oligonucleotide ("tag molecule") so that it forms a tag with the tag oligonucleotide in a second set of conditions ("tag molecule with the tag of the hairpin "). If it is part of a tag molecule, the tag closing sequence is preferably linked to the tagged oligonucleotide via a non-nucleotide linker region (e.g. baseless nucleotides or polyethylene glycol) sufficiently long for the tag closing sequence to hybridize to the target hybridizing sequence under the second set of conditions. The tag closing sequence may be modified to prevent DNA synthesis from beginning therefrom, which may include a blocking moiety located at its 3'-end. The tag closing sequence is at least 3 but not more than about 20 bases in length. Typical tag closing sequences are 10 to 16 bases long.

Nucleic acid amplification or amplification [0135] By "amplification" or "nucleic acid amplification" is meant the production of multiple copies of a target nucleic acid that contain at least a fragment of the desired specific target nucleic acid sequence. These numerous copies may be called amplicons or amplification products. In certain embodiments, the amplified target contains less than the complete target gene sequence (introns and exons) or the expressed target gene sequence (spliced ​​transcript consisting of exons and flanking non-translated sequences). For example, specific amplicons can be produced by amplifying a fragment of the target polynucleotide by using amplification primers that hybridize and start polymerization from internal positions in the target polynucleotide. Preferably, the amplified fragment comprises a detectable target sequence that can be detected using any of a variety of well-known methods.

[0136] Many well-known nucleic acid amplification methods require cyclic temperature changes for alternating denaturation of double-stranded nucleic acids and hybridization of primers; however, other well known methods of nucleic acid amplification are isothermal. Polymerase chain reaction (Mullis et al., U.S. Patent No. 4,681,195; Mullis, U.S. Patent No. 4,683,202; and Mullis et al., U.S. Patent No. 4,800,159), commonly referred to as PCR, uses numerous denaturation cycles, steam attachment reverse strand primers and extension primers to exponentially increase the number of copies of the target sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to make complementary DNA (cDNA) from mRNA, and then the cDNA is amplified by PCR to generate multiple copies of DNA (Gelfand et al., "Reverse Transcription with Thermostable DNA Polymerases High Temperature Reverse Transcription ", US Patent Nos. 5,322770 and 5310652). Thread displacement amplification is another method (Walker, G. et al. (1992), Proc. Natl. Acad. Sci. USA 89, 392-396; Walker et al., "Nucleic Acid Target Generation," US Patent No. 5,270,184; Walker, "Strand Displacment Amplification", US Patent No. 5,455,166; and Walker et al. (1992) Nucleic Acids Research 20, 1691-1696), commonly known as SDA, which uses cycles to attach primer sequence pairs to opposite strands of the target sequence, primer extension in the presence of dNTP to produce a hemithiophosphorylated primer extension product, endonuclease-mediated incision of hemi-modified endonuclease-recognized restriction sites and polymerase-mediated primer extension from the 3 'end of the incision to displace the existing strand and create the thread for the next round of primer attachment, cut and displace strand, resulting in geometric amplification of the product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same way (European Patent No. 0 684 315). Other amplification methods include: nucleic acid sequence based amplification (Malek et al., U.S. Patent No. 5,130,238), commonly referred to as NASBA; one that uses RNA replicase to amplify the probe molecule itself (Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202), commonly called Qe replicase; transcription based amplification method (Kwoh, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173-1177); self-sustaining sequence replication (Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199202; and Lee, H. et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); and transcription-mediated amplification (Kacian et al., "Nucleic Acid Sequence Amplification Methods", US Patent No. 5,480,784; and Kacian et al., US Patent No. 5,399,491), commonly referred to as TMA. For further discussion of known amplification methods, see Persing, David H., 1993, "In Vitro Nucleic Acid Amplification Techniques" in Diagnostic Medical Microbiology: Principles and Applications (Persing et al., Ed.), P. 51-87 (American Society for Microbiology, Washington, DC). Other exemplary amplification methods convenient for use in accordance with the present invention include "rolling circle" (RCA) amplification (Lizardi, "Rolling Circle Replication Reporter Systems", US Patent No. 5,854033); helicase-dependent (HDA) amplification (Kong et al., "Helicase Dependent Amplification Nucleic Acids", US Patent Application Publication No. US 2004-0058378 A1); and isothermal loop-mediated amplification (LAMP) (Notomi et al., "Process for Synthesizing Nucleic Acid", US Patent No. 6410278).

[0137] Preferred transcription-based amplification systems of the present invention include TMA, which uses RNA polymerase to generate multiple RNA transcripts of the target region (e.g., Kaoian et al., U.S. Patent Nos. 5480784 and 5399491; and Becker et al., "Single-Primer Nucleic Acid Amplification Methods", US Patent Application Publication No. US 2006-0046265 A1). TMA uses a "promoter oligonucleotide" or "promoter-primer" that hybridizes to the target nucleic acid in the presence of reverse transcriptase and RNA polymerase to form a double-stranded promoter from which the DNA polymerase produces RNA transcripts. These transcripts may become templates for subsequent TMA rounds in the presence of a second primer capable of hybridizing to RNA transcripts. Unlike PCR, LCR or other methods that require high temperature denaturation, TMA is an isothermal method that uses RNAse H activity to digest RNA strands from an RNA: DNA hybrid, thereby making the DNA strand available for hybridization with a primer or promoter-primer. Typically, RNAse H activity associated with reverse transcriptase delivered for amplification is used.

[0138] In one exemplary TMA method, one amplification primer is an oligonucleotide promoter-primer that includes a promoter sequence that becomes functional when double-stranded 5 'to the target binding sequence that is capable of hybridizing to a binding site target RNA located 3 'to the sequence to be amplified. The promoter-primer may be called "T7 primer" when it is specific for recognition by T7 RNA polymerase. Under certain circumstances, the 3'-end of the promoter-primer, or subpopulation of such promoter-primers may be modified to block or limit primer extension. Starting with the unmodified promoter-primer, reverse transcriptase creates cDNA that is a copy of the target RNA, while RNAse H activity degrades the target RNA. The second amplification primer then binds to this cDNA. This primer may be called a "non-T7 primer" to distinguish it from a "T7 primer". Starting from this second amplification primer, reverse transcriptase forms a second DNA strand, resulting in double-stranded DNA with a functional promoter at one end. When double-stranded, the promoter sequence is capable of binding RNA polymerase to initiate transcription of the target sequence with which the promoter-primer is hybridized. RNA polymerase uses this promoter sequence to generate multiple RNA transcripts (i.e., amplicons), generally about 100 to 1000 copies. Each newly synthesized amplicon may associate with a second amplification primer. Reverse transcriptase can then produce a copy of DNA, while RNAse H activity degrades RNA from this RNA: DNA duplex. The promoter-primer can then bind to newly synthesized DNA, allowing reverse transcriptase to produce double-stranded DNA from which RNA polymerase forms many amplicons. One billion times isothermal amplification can be achieved using two amplification primers.

[0139] In another exemplary TMA method, one or more of the properties described in Becker et al., US Patent Application Publication No. US 2006-0046265 A1, can optionally be incorporated. Preferred TMA methods in this regard include the use of blocking moieties, terminating moieties and other modifying moieties that provide improved sensitivity and accuracy of the TMA process. Thus, in certain preferred embodiments of the present invention, labeled oligonucleotides as described herein are used in conjunction with the methods described in Becker et al., US Patent Application Publication No. US 2006-0046265 A1.

[0140] By "detectable amplification" is meant that the detectable signal associated with the amplification product in the amplification reaction mixture rises above a predetermined background or threshold level (endpoint amplification) or rises above a background or threshold level for a predetermined period of time (real-time amplification). See e.g. Light et al., "Method for Determining the Amount of an Analyte in a Sample", US Patent Application Publication No. US 2006-0276972, paragraphs 506-549. The amplification product contains a sequence with sequence identity to the target nucleic acid sequence or its complement and can be detected, for example using an intercalating dye or detection probe specific for the region of the target nucleic acid sequence or its complement.

"Selective amplification" [0141] The term "selective amplification" as used herein refers to the amplification of a target nucleic acid sequence according to the present invention in which the detectable amplification of the target sequence is limited or substantially limited to the amplification of the target sequence provided in the test sample , and is not derived from the target nucleic acid sequence provided in the sample from some other source, e.g. contamination present in the reagents or components used during the amplification reaction or in the environment or environmental conditions in which the amplification reactions are carried out.

Amplification Conditions [0142] By "amplification conditions" is meant conditions that allow nucleic acid amplification according to the present invention. Amplification conditions may in some embodiments be less stringent than "stringent hybridization conditions" as described herein. The oligonucleotides used in the amplification reactions of the present invention hybridize to their intended targets under amplification conditions, and may or may not hybridize under stringent hybridization conditions. On the other hand, the detection probes of the present invention hybridize under stringent hybridization conditions. While the following section of the Examples provides favorable amplification conditions for the amplification of target nucleic acid sequences in accordance with the present invention, other acceptable conditions for performing nucleic acid amplification in accordance with the present invention can be easily determined by one of ordinary skill in the art depending on the particular method used for amplification.

Hybridization / hybridization [0143] Nucleic acid hybridization is a process in which two nucleic acid strands with completely or partially complementary nucleotide sequences assemble together under predetermined reaction conditions to form a stable double-stranded hybrid. Each nucleic acid strand may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or analogues thereof. Thus, hybridization may include RNA: RNA hybrids, DNA: DNA hybrids, RNA: DNA hybrids or analogs thereof. These two threads that make up this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. Although these hydrogen bonds are most commonly formed between nucleotides containing bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on single nucleic acid strands, base pairing may also occur between non-base bases of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, e.g., ROGER LP ADAMS AND OTHERS, THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (11th edition, 1992)).

[0144] "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which a specific detection probe is capable of hybridizing to target nucleic acids among other nucleic acids present in the test sample. It will be understood that these conditions may vary depending on various factors, including GC content and probe length, hybridization temperature, composition of reagents or hybridization solutions, and the expected degree of hybridization specificity. Specific stringent hybridization conditions are provided in the disclosure below.

[0145] By "hybrid nucleic acid" or "hybrid" or "duplex" is meant a nucleic acid structure comprising a double-stranded hydrogen bonded region in which each strand is complementary to the other, and which region is sufficiently stable under stringent conditions hybridization so that it can be detected by methods such as, but not limited to, detection of chemiluminescent or fluorescent light, autoradiography or gel electrophoresis. Such hybrids may include RNA: RNA, RNA: DNA or DNA: DNA duplex molecules.

[0146] By "complementary" is meant that nucleotide sequences from similar regions of two single-stranded nucleic acids or from two different regions of the same single-stranded nucleic acid have a nucleotide base composition that allows single-stranded regions to hybridize to each other in stable double-stranded linkages hydrogen region under stringent hybridization or amplification conditions. When a continuous nucleotide sequence from one single-stranded region is capable of forming a series of "canonical" hydrogen bonded base pairs with an analogous sequence of nucleotides from the other single-stranded region, so that A is paired with U or T, and C is paired with G, these nucleotide sequences are "perfectly" complementary.

[0147] By "hybridize preferentially" is meant that under stringent hybridization conditions, specific complementary nucleotide or nucleic base sequences hybridize to form a stable hybrid preferentially over other less stable duplexes. By "do not stably hybridize" is meant that a stable hybrid is not formed in significant and / or detectable amounts under a defined set of conditions.

[0148] By "stable" or "stably hybridize" is meant that the temperature of the reaction mixture is at least 2 ° C lower than the melting point of the nucleic acid duplex.

Promoter oligonucleotide / promoter sequence [0149] As is well known in the art, "promoter" is a specific nucleic acid sequence that is recognized by DNA-dependent RNA polymerase ("transcriptase") as a signal to bind to nucleic acid and initiate RNA transcription at a specific site . It was generally believed that such transcriptases required DNA that became double-stranded in the region containing the promoter sequence in the extension reaction, however, the present inventors have found that effective RNA transcription can occur even under conditions in which the double-stranded promoter is not formed in the extension reaction from matrix nucleic acid. The template nucleic acid (sequence to be transcribed) need not be double-stranded. Individual DNA-dependent RNA polymerases recognize many different promoter sequences that can be clearly different in their transcriptional efficiency. When RNA polymerase binds to the promoter sequence to initiate transcription, this promoter sequence is not part of the transcribed sequence. Thus, RNA transcripts produced in this way will not contain this sequence.

[0150] In accordance with the present invention, "promoter oligonucleotide" refers to an oligonucleotide comprising the first and second regions, and which is preferably modified to prevent DNA synthesis from starting at its 3'-end. The "first region" of the promoter oligonucleotide includes a base sequence that hybridizes to the DNA template, wherein the hybridizing sequence is 3 'to the promoter region, but does not necessarily adhere to it. The hybridizing promoter oligonucleotide fragment of the present invention is typically at least 10 nucleotides long, and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. The "second region" includes the promoter for RNA polymerase. The promoter oligonucleotide of the present invention is designed so that it is not capable of being extended by RNA or DNA dependent DNA polymerase, e.g. reverse transcriptase, preferably having a blocking moiety at its 3'-end as described above. Convenient and preferred promoter oligonucleotides are described herein.

Universal / global oligonucleotides [0151] "Universal" oligonucleotides or "global" oligonucleotides include oligonucleotides that can be used in an amplification reaction to identify the presence of nucleic acid sequences from a class of organisms based on highly conserved sequences that are unique to this class of organisms. (The term "class" does not necessarily mean known phylogenetic groups or organisms as used herein.) For example, highly conserved coding sequences for 16S ribosomal RNA contain regions that can be found in bacteria or bacterial groups (e.g., specific bacteria, Gram-positive bacteria, or Gram-negative bacteria), but are not found in humans and other higher organisms, but thus, oligonucleotides can be designed and used in a nucleic acid amplification reaction to detect the presence of bacterial sequences in the sample of interest. See, e.g., McCabe et al. (1999) Molecular Genetics and Metabolism 66, 205-211; Schmidt, T. et al. (1994) Meth. Enzymol. 235, 20548

222 (method of identifying pathogens); Kunishima, S. et al. (2000) Transfusion 40, 1420 (method for detecting bacteria in the blood); Greisen, K. (1994) J. Clin. Microbiol. 32, 335-351 (method for detecting pathogenic bacteria in cerebrospinal fluid); Jordan, J. (2005) J. Mol. Diag. 7, 575-581 (method of diagnosis of sepsis in newborns); Rothman, R. et al. (2002) J. Infect. Dis. 186, 1677-1681 (method for the diagnosis of acute bacterial endocarditis); and Cox, C. et al. (2002) Arthritis Res. Ther. 5, R1-R8 (detection of bacteria in synovial fluid). Similarly, universal oligonucleotides for other classes of organisms, such as fungal pathogens, have been described. See, e.g., Maaroati, Y. et al. (2003) J. Clin. Microbiol. 41, 3293-3298 (method for quantifying blood Candida albicans); Carr, M. et al. (2005) J. Clin. Microbiol. 43, 3023-3026 (method for detecting Candida dubliniensis in the blood); and White, P. et al. (2003) J. Med Microbiol. 52, 229-238 (method for detecting systemic fungal infections). In principle, any universal oligonucleotide known or produced for a given class of organisms can be advantageously used in the methods described herein.

The priming oligonucleotide [0152] The priming oligonucleotide is an oligonucleotide whose at least 3'-end is complementary to the nucleic acid matrix and which enters the complex (through hydrogen bonding or hybridization) with the matrix to form a starter: matrix template suitable for the start of synthesis by dependent DNA polymerase from RNA or DNA. The priming oligonucleotide is extended by attaching covalently linked nucleotide bases to its 3'-end, which bases are complementary to the template. The result is a primer extension product. The priming oligonucleotide of the present invention is usually at least 10 nucleotides in length and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Convenient and preferred primer oligonucleotides are described herein. Virtually all known DNA polymerases (including reverse transcriptases) require the formation of an oligonucleotide complex with a single-stranded matrix ("priming") to start DNA synthesis, while RNA replication and transcription (copying of RNA from DNA) generally requires no primer. Due to the characteristic feature of the priming oligonucleotide, which is extension by DNA polymerase, it does not include a 3'-blocking moiety.

Displacement oligonucleotide [0153] The term "displaceable oligonucleotide" means a priming oligonucleotide that hybridizes to a template nucleic acid upstream of an adjacent priming oligonucleotide hybridized to the 3'-end of the target sequence (referred to herein as the "primer oligonucleotide" herein). By 'up' it is meant that

The 3'-end of the displaceable oligonucleotide enters the complex with the template nucleic acid in the 5 'to 3'-end direction of the forward priming oligonucleotide. When hybridized to the template nucleic acid, the 3'-terminal displacement oligonucleotide base preferably adheres to or is separated from the 5-terminal base of the priming oligonucleotide. More preferably, the 3'-terminal base of the displacer oligonucleotide is separated by 5 to 35 bases from the 5'-base of the forward primer oligonucleotide. The displacer oligonucleotide may be delivered to the reaction mixture simultaneously with the forward primer oligonucleotide or after the forward primer oligonucleotide has already had sufficient time to hybridize with the template nucleic acid. Forward elongation of the primer oligonucleotide can be started before or after delivery of the displacement oligonucleotide to the reaction mixture. Under amplification conditions, the displacer oligonucleotide is extended in a matrix-dependent manner, thereby displacing the primer extension product comprising the forward primer oligonucleotide that forms a complex with the template nucleic acid. After displacement from the template nucleic acid, a primer extension product comprising a forward primer oligonucleotide is available to form a complex with the promoter oligonucleotide. Both the forward primer oligonucleotide and displacement oligonucleotide preferentially hybridize with the target nucleic acid. Examples of displacement oligonucleotides and their uses are disclosed by Becker et al., "Methods and Kits for Amplifying DNA", US Patent Application No. 11 / 681,104, on behalf of the same applicant as the present application.

Blocking moiety [0154] As used herein, the term "blocking moiety" means a substance used to "block" the 3'-end of an oligonucleotide or other nucleic acid such that it cannot be effectively extended by nucleic acid polymerase. The blocking moiety may be a small molecule, e.g. a phosphate or ammonium group, or may be a modified nucleotide, e.g. 3 ', 2'-dideoxynucleotide or 3'-deoxyadenosine 5'-triphosphate (cordycepine) or other modified nucleotide. Additional blocking moieties include, for example, the use of a nucleotide or a short sequence of nucleotides with a 3'-to-5 'orientation such that there is no free hydroxyl group at the 3'-end, the use of a 3'-alkyl group, a 3'-non-nucleotide moiety (see e.g. . Arnold et al., "Non-Nucleotide Linking Reagents for Nucleotide Probes," US Patent No. 6031091), phosphorothioate, alkanediol residues, peptide nucleic acid (PNA), nucleotide residues lacking a 3'-hydroxyl group at the 3'-end or protein nucleic acid binding. Preferably the 3'-blocking moiety comprises a nucleotide or nucleotide sequence with a 3'-to-5 'orientation or a 3'-non-nucleotide orientation, and not a 3', 2'-dideoxynucleotide or a 3'-end with a free hydroxyl group. Additional methods for preparing 3'-blocking oligonucleotides are well known to those of ordinary skill in the art.

Binding molecule [0155] As used herein, the term "binding molecule" means a substance that hybridizes or otherwise binds to a target RNA nucleic acid adjacent to or near the 5'-end of the desired target sequence so as to limit the DNA product extension a primer to the desired length, i.e. a primer extension product with a generally defined 3'-end. As used herein, the expression "defined 3'-end" means that the 3'-end of the primer extension product is not completely indeterminate, as would be the case with the primer extension reaction that occurs in the absence of a binding molecule, but rather that the 3'- the end of the primer extension product is generally known within a small range of bases. In certain embodiments, the binding molecule comprises a base region. The base region may be DNA, RNA, a DNA: RNA chimeric molecule or an analogue thereof. Binding molecules containing a base region can be modified in one or more ways as described herein. Exemplary base regions include terminating and digesting oligonucleotides as described below. In other embodiments, the binding molecule may include, for example, a protein or drug capable of binding RNA with sufficient affinity and specificity to limit the DNA primer extension product to a predetermined length.

The terminating oligonucleotide [0156] In the present invention, the term "terminating oligonucleotide" means an oligonucleotide comprising a base sequence that is complementary to a region in the target nucleic acid around the 5'-end of the target sequence so as to "complete" primer extension in the resulting nucleic acid that includes a priming oligonucleotide, thereby providing a defined 3'-end of the resulting nucleic acid strand. The terminating oligonucleotide is designed to hybridize to the target nucleic acid in a position sufficient to obtain the desired 3 'end of the resulting nucleic acid strand. The position of the terminating oligonucleotide is flexible depending on its design. The terminating oligonucleotide can be modified or unmodified. In certain embodiments, terminating oligonucleotides are synthesized from at least one or more 2'-O-ME ribonucleotides. These modified nucleotides showed higher thermal stability of complementary duplexes. The function of 2'-O-ME-ribonucleotides is also to increase the resistance of oligonucleotides to exonucleases, thereby increasing the half-life of modified oligonucleotides. See, e.g., Majlessi et al. (1988) Nucleic Acids Res. 26, 2224-9. Other modifications, as described elsewhere herein, may be used together with or instead of 2'-OME ribonucleotides. For example, the terminating oligonucleotide may comprise PNA or LNA. See, e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53. The terminating oligonucleotide of the present invention typically includes a blocking moiety at its 3'-end to prevent elongation. The terminating oligonucleotide may also include a protein or peptide attached to the oligonucleotide so as to complete the further extension of the resulting nucleic acid chain by the polymerase. The terminating oligonucleotide of the present invention is usually at least 10 bases long, and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Convenient and preferred oligonucleotides are described herein. It should be noted that while the terminating oligonucleotide usually or necessarily includes a 3'-blocking moiety, "3'-blocked" oligonucleotides are not necessarily terminating oligonucleotides. Other oligonucleotides of the present invention, e.g. promoter oligonucleotides and capped oligonucleotides are also usually or necessarily 3'-blocked.

Inserted sequence [0157] As used herein, an "inserted sequence" is a sequence located between the first region (ie, the template binding fragment) and the second promoter oligonucleotide region. The inserted sequences are preferably 5 to 20 nucleotides in length, more preferably 6 to 18 nucleotides in length, and most preferably 6 to 12 nucleotides in length. Incorporation of inserted sequences into promoter oligonucleotides increases the speed at which RNA amplification products are created. Exemplary inserted sequences are described herein.

Targeted Uptake [0158] As used herein, targeted uptake includes any effective technique to remove all or substantially all of the unhybridized tagged oligonucleotide after hybridizing the tagged oligonucleotide to the target nucleic acid sequence but prior to amplifying the target nucleic acid sequence. Typically, targeted uptake involves capturing the target polynucleotide on a solid support, such as magnetically attracting particles, said solid support retaining the target polynucleotide during one or more wash steps from the purification procedure of the target polynucleotide. In this way, the target polynucleotide is substantially purified from unhybridized tagged oligonucleotide prior to the next nucleic acid amplification step. Numerous targeted uptake methods are known and convenient for use in combination with the methods described herein.

[0159] For example, in one illustrative approach described in US Patent Application Publication No. US 2006-0068417 A1, at least one oligonucleotide is a capture probe that comprises a target complementary region and a member of a specific binding pair that connects the target nucleic acid to the immobilized probe on the capture substrate, thereby forming a capture hybrid that is separated from the other components of the sample from the sample. In another exemplary method, Weisburg et al., U.S. Patent No. 6,110,678, describes a method of capturing a polynucleotide target in a sample on a solid support, such as a magnetically attracting particle, with an attached immobilized probe by using a capture probe and two different sets of hybridization conditions that they preferably only differ in temperature. These two sets of conditions control the order of hybridization, with the first hybridization conditions enabling the capture probe to hybridize with the target polynucleotide, and the second hybridization conditions enabling the capture probe to hybridize with the immobilized probe. This method can be used to detect the presence of a target polynucleotide in a sample by detecting the captured target polynucleotide or amplified target polynucleotide.

[0160] Another exemplary targeted capture technique includes a sandwich hybridization technique for capture and detection of the presence of the target polynucleotide. See Ranki et al., "Detection of Microbial Nucleic Acids By a One-Step Sandwich Hybridization Test", US Patent No. 4,486,539. These techniques include uptake of the target polynucleotide by a probe bound to the solid support and hybridizing the detection probe to the captured target oligonucleotide. Detection probes not hybridized to the target polynucleotide are easily washed away from the solid support. Thus, the remaining tag is associated with the target polynucleotide initially present in the sample.

[0161] Another exemplary targeted capture technique involves a method that uses an intermediary polynucleotide that hybridizes to both the target polynucleotide and the polynucleotide attached to a solid support. See Stabinsky, "Methods and Kits for Performing Nucleic Acid Hybridization Assays", US Patent No. 4,751,177. An intermediary polynucleotide combines a target polynucleotide with a solid support to form a bound target. The labeled probe can be hybridized to the bound target, and the unbound labeled probe can be flushed out of the solid support.

[0162] Yet another exemplary directed capture technique is disclosed by Englelhardt, "Ca pture Sandwich Hybridization Method and Composition", US Patent No. 5,288,609, which describes a method of detecting a target polynucleotide. This method uses two single-stranded polynucleotide segments complementary to the same or opposite strands of the target and produces a double-stranded hybrid with the target polynucleotide. In one embodiment, the hybrid is captured on a solid support.

[0163] In another exemplary directed capture technique, oligonucleotide primers labeled with specific binding partners are used in methods and kits for detecting nucleic acids to immobilize primers and primer extension products. See Burdick et al., "Diagnostic Kit and Method Using a Solid Phase Capture Means for Detecting Nucleic Acids", European Patent Application No. 0 370 694 A2. The label specifically forms a complex with its receptor, which is bound to a solid support.

[0164] The above capture techniques are merely exemplary and not limiting. In fact, any technique available to a person skilled in the art can be used, provided that it is effective in removing all or substantially all of the unhybridized tagged oligonucleotide after hybridizing the tagged oligonucleotide to the target nucleic acid sequence but prior to amplifying the target nucleic acid sequence as described herein.

Probe [0165] By "probe" or "detection probe" is meant a molecule comprising an oligonucleotide with a base sequence partially or completely complementary to a region from the target sequence to be detected so that it hybridizes to it under stringent hybridization conditions. As will be understood by one of ordinary skill in the art, the probe includes an isolated nucleic acid or analogue molecule in a form not found in nature without human intervention (e.g. recombinant with a foreign nucleic acid, isolated or to some extent purified).

[0166] The probes of this invention may have additional nucleosides or nucleobases outside the target region, provided that such nucleosides or nucleobases do not significantly affect hybridization under stringent hybridization conditions and, in the case of detection probes, do not prevent preferential hybridization with the target nucleic acid . An incomplete sequence may also be included, such as a target capture sequence (generally a homopolymeric sequence such as poly-A, poly-T or poly-U tail), promoter sequence, RNA transcription binding site, endonuclease-recognized restriction site or sequences, which will give the probe, target nucleic acid, or both, the desired secondary or tertiary structure, such as a catalytically active site or hairpin structure.

[0167] The probes preferably comprise at least one detectable label. The label can be any labeling substance, including, but not limited to, a radioisotope, enzyme, enzyme cofactor, enzyme substrate, dye, hapten, chemiluminescent molecule, fluorescent molecule, phosphorescent molecule, electrochemiluminescent molecule, chromophore, base sequence region that is unable to stable hybridization to the target nucleic acid under determined conditions, and mixtures thereof. In one particularly preferred embodiment, the label is an acridine ester. Probes may also include interactive labels that emit different signals depending on whether the probes have hybridized to the target sequences. Examples of interactive labels include enzyme / substrates, enzyme / cofactor, luminescent compound / quencher, luminescent compound / adduct, dye dimers and Fórrester's energy transfer pairs. Certain probes of the present invention do not include a label. For example, unlabeled "capture" probes can be used to enrich for target sequences or their replicons, which can then be detected by a second "detection" probe. See, e.g., Weisburg et al., US Patent No. 6,534,273. While detection probes are typically labeled, specific detection technologies do not require the probe to be labeled. See, e.g., Nygren et al., "Devices and Methods for Optical Detection of Nucleic Acid Hybridization", US Patent No. 6060237.

[0168] By "stable" or "stable for detection" is meant that the temperature of the reaction mixture is at least 2 ° C lower than the melting point of the nucleic acid duplex. The temperature of the reaction mixture is more preferably at least 5 ° C lower than the melting point of the nucleic acid duplex, and even more preferably at least 10 ° C lower than the melting point of the reaction mixture.

[0169] By "hybridization preferentially" is meant that under stringent hybridization conditions, the probes of the present invention hybridize to their target sequences or their replicates to form stable probe: target hybrids, while at the same time the formation of stable hybrids: probe: molecule other than goal. Thus, the probe hybridizes to the target sequence or its replica to a sufficiently higher degree than to the non-target sequence, enabling the average skilled person to accurately quantify the RNA replicas or complementary DNA (cDNA) of the target sequences generated during amplification.

[0170] Probes with a defined sequence can be prepared by techniques known to those of ordinary skill in the art, such as chemical synthesis and in vitro or in vivo expression from recombinant nucleic acid molecules. Preferably the probes are from 10 to 100 nucleotides in length, more preferably 12 to 50 bases, and even more preferably 18 to 35 bases.

Nucleic acid "identity" [0171] In certain embodiments, the nucleic acid of the present invention comprises a region of contiguous bases that is at least 80%, 90% or 100% identical to the adjacent base region of the reference nucleic acid. For short nucleic acids, e.g. of specific oligonucleotides of the present invention, the degree of identity between the base region of the "query" nucleic acid and the base region of the reference nucleic acid can be determined by manual alignment. "Identity" is determined by comparing only nitrogen base sequences, regardless of the sugar regions and backbone of the compared nucleic acids. Thus, the base sequence query: reference can be DNA: DNA, RNA: RNA, DNA: RNA, RNA: DNA, or any combinations or analogues thereof. Equivalent RNA and DNA base sequences can be compared by converting all U (in RNA) to T (in DNA).

Matrix [0172] The term "matrix" means a nucleic acid molecule that is copied by a nucleic acid polymerase. The matrix can be single-stranded, double-stranded or partially double-stranded, depending on the polymerase. The synthesized copy is complementary to the matrix or at least one thread of a double-stranded or partially double-stranded matrix. Both RNA and DNA are usually synthesized in the 5'to-3 'direction, and the two strands of the nucleic acid duplex are positioned so that the 5' ends of these two strands are at opposite ends of the duplex (then just as necessary, ends 3 '). While in accordance with the present invention, the "target sequence" is always a "template", templates can also include secondary primer extension products and amplification products.

DNA dependent DNA polymerase [0173] The term "DNA dependent DNA polymerase" means an enzyme that synthesizes a complementary copy of DNA from a DNA template. Examples are Taq DNA polymerase, highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus, for PCR amplification reactions, DNA polymerase I from E. coli, DNA polymerase from bacteriophage T7, or DNA polymerase from bacteriophages T4, Phi-29, M2 or T5. The DNA-dependent DNA polymerases of the present invention may be naturally occurring enzymes isolated from bacteria or bacteriophages or produced by recombinant expression, or may be modified or "evolved" forms that have been designed to have specific desired characteristics, e.g., thermostability or the ability to recognize or synthesize DNA strands from various modified templates. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. It is known that under appropriate conditions DNA-dependent DNA polymerase can synthesize a complementary copy of DNA from an RNA template. RNA-dependent DNA polymerases (described below) also typically have DNA-dependent DNA polymerase activity. An example of such a polymerase is Tth MasterAmp ™ DNA Polymerase, which has both DNA-dependent and RNA-dependent DNA polymerase activity (i.e. reverse transcriptase) that can be used in both PCR and RT-PCR amplification reactions (Epicenter Biotechnologies, Madison, WI).

DNA-dependent RNA polymerase (Transcriptase) [0174] The term "DNA-dependent RNA polymerase" means an enzyme that synthesizes multiple RNA copies from a double-stranded or partially double-stranded DNA molecule having a promoter sequence, which is usually double-stranded. RNA molecules ("transcripts") are synthesized in the 5'-to-3 'direction starting from the specific sequence just down from the promoter. Examples of transcriptases are DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, and SP6.

RNA-dependent DNA polymerase (reverse transcriptase) [0175] The term "RNA-dependent DNA polymerase" or "reverse transcriptase" ("RT") means an enzyme that synthesizes a complementary copy of DNA from an RNA template. All known reverse transcriptases also have the ability to make a complementary copy of DNA from a DNA template; are therefore both DNA RNA and DNA polymerase. RT may also have RNAse H activity. Reverse transcriptase derived from the Maloney mouse leukemia virus (MMLV-RT) is preferred. A primer is required for both RNA and DNA templates to start the synthesis.

Selective RNAse [0176] As used herein, the term "selective RNAse" means an enzyme that degrades an RNA fragment from an RNA duplex: DNA, but not single-stranded RNA, double-stranded RNA or DNA. An example of selective RNAse is RNAse H. Enzymes other than RNAse H that have the same or similar activity are also contemplated in the present invention. Selective RNAse may be endonucleases or exonucleases. Most reverse transcriptase enzymes have RNAse H activity outside of their polymerase activity. However, other RNAz H sources are available with no associated polymerase activity. Degradation may result in the separation of RNA from the RNA: DNA complex. Alternatively, selective RNAse may simply cleave the RNA at various locations, so that RNA fragments are rendered or allow the enzymes to develop RNA fragments. Other enzymes that selectively degrade RNA target sequences or RNA products of the present invention will be clearly visible to those of ordinary skill in the art.

Sense / Antisense Strand (s) [0177] Discussions of nucleic acid synthesis are very simplified and clarified by adopting terms to name two complementary strands from a nucleic acid duplex. Traditionally, the strand encoding the sequences used to produce proteins or structural RNAs is designated as the "sense (+)" strand and its complement as "antisense (-)" strand. It is now known that in many cases both threads are functional, and the assignment of the designation "sensible" to one and "antisense" to the other must then be optional. Still, these terms are very useful for determining sequence orientation in nucleic acids and will be used here for this purpose.

System specificity [0178] The term "specificity" in the context of an amplification system is used herein to refer to the properties of an amplification system that describes its ability to distinguish between target and non-target sequences depending on the sequence and test conditions. With respect to nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of by-products (i.e. signal-to-noise ratio), as described in more detail below.

Sensitivity [0179] The term "sensitivity" is used herein to refer to the accuracy with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of the amplification reaction is generally a measure of the smallest number of copies of the target nucleic acid that can be reliably detected in an amplification system, and will depend, for example, on the detection test used and the specificity of the amplification reaction, i.e. ratio of specific amplicons to by-products.

Bioprocess [0180] The term "bioprocess" as used herein generally refers to any process in which live cells or their components are intentionally or unintentionally present. For example, in principle, any production or other process that uses one or more samples or sample streams, at least one of which contains live cells or their components, or may contain such cells or components as a result of unintentional contamination, is considered a bioprocess . In many such processes, it is desirable to have the ability to detect, identify and / or control the presence and / or sources of live cells and their components within the process. Using the methods of the present invention, for example, the presence and / or sources of contaminating microorganisms or other biological material or components thereof can be monitored in one or more bioprocess samples or streams. In addition, purification / sterilization requirements in specific samples / bioprocess streams can be advantageously reduced using the methods of the invention as set forth herein.

[0181] As discussed above, the present invention relates generally to nucleic acid amplification methods that preferably limit or eliminate false positive amplification signals resulting from a contaminating biological material, such as nucleic acid material, which may be present in one or more reagents , samples or components used in the amplification reaction or which may be present in the environment, in which the amplification reactions are carried out. The invention further provides the advantage of requiring less stringent purification and / or sterility procedures than the standard required to ensure that enzymes and other reagents and components used in amplification reactions and the environment in which the amplification reactions are carried out are free of bacteria and other nucleic acid contaminants which may give false positive results. Accordingly, the methods of the invention are particularly useful in detecting, monitoring and / or quantifying microorganisms (or contaminating nucleic acids from other sources) in clinical samples, bioprocess samples or sample streams, food products, water, industrial and environmental samples, inventory seeds and other types of material, in which the detection and / or monitoring of the presence of microorganisms or other forms of contamination may be required.

[0182] The present invention may be adapted for use in virtually any amplification procedure requiring a primer oligonucleotide that binds to the matrix and is capable of being extended in the presence of nucleic acid polymerase. Incorporation of labeled oligonucleotides (or heterologous tag sequences) into such primer-dependent amplification procedures can be achieved without significant modification of the reagents and reaction conditions of such procedures. Any necessary modifications should be minimal and will be within the knowledge and skills of an experienced molecular biologist. Descriptions of various exemplary amplification procedures using labeled oligonucleotides are provided below.

[0183] FIG. 1 illustrates the adaptation of an isothermal transcription-based amplification reaction known as reverse transcription-mediated amplification (rTMA), various aspects of which are disclosed in Becker et al., US Patent Application Publication No. US 20060046265 A1. The reaction of this exemplary embodiment is initiated by treating the target RNA sequence in a nucleic acid sample with both a labeled priming oligonucleotide and a terminating oligonucleotide. The tagged priming oligonucleotide comprises a target hybridizing sequence that hybridizes to the 3'-end of the target sequence and a tag sequence located 5 'to the target hybridizing sequence. The terminating oligonucleotide hybridizes to the target nucleic acid containing the target sequence near the 5'-end of the target sequence. The terminating oligonucleotide is used to terminate primer extension in a nascent nucleic acid that includes a tagged priming oligonucleotide. Thus, the target nucleic acid forms a stable complex with the tagged priming oligonucleotide at the 3'-end of the target sequence and the terminating oligonucleotide located adjacent to or near the 5'-end of the target sequence before starting the primer extension reaction. See FIG. 1, Stage 1. The unhybridized tagged priming oligonucleotide is made unavailable for hybridization with the target sequence prior to starting the primer extension reaction from the tagged priming oligonucleotide, preferably by inactivating and / or removing the unhybridized tagged priming oligonucleotide from the nucleic acid sample.

[0184] Then, from the 3'-end of the tagged priming oligonucleotide, a DNA polymerase extension reaction, e.g., reverse transcriptase, begins to form the first DNA primer extension product that includes the tag sequence and region complementary to the target sequence. See FIG. 1, Steps 2 and 3. The first primer extension product is then separated from the target sequence using an enzyme that selectively degrades the target sequence (e.g., with RNAse H activity). See FIG. 1, Stage 4.

[0185] The first DNA primer extension product is then treated with a promoter oligonucleotide comprising a hybridizing sequence and an RNA polymerase promoter located 5 'to the hybridizing sequence. The hybridizing sequence hybridizes to the region of the first DNA primer extension product that is complementary to the 3'-end of the target sequence, thereby forming a hybrid promoter oligonucleotide: the first DNA primer extension product. In the illustrated reaction, the promoter oligonucleotide is modified to prevent the initiation of DNA synthesis, preferably by placing a blocking moiety at the 3'-end of the promoter oligonucleotide (e.g., 3'-to-5 'nucleotide sequence). See FIG. 1, Step 5. The 3'-end of the first DNA extension product is preferably extended to attach the complementary sequence to the promoter, resulting in the formation of a double-stranded promoter sequence. See FIG. 1, Stages 6 and 7. Numerous copies of the first RNA product complementary to at least part of the first DNA primer extension product, not including the promoter fragment, are then transcribed using RNA polymerase, which recognizes the double-stranded promoter and starts transcribing from it. See FIG. 1, Steps 8 and 9. As a result, the base sequence of the first RNA product is substantially identical to the base sequence of the target sequence and the complement of the tag sequence.

[0186] The first RNA products are treated with a priming oligonucleotide that hybridizes to the complement of the tag sequence to form a hybrid priming oligonucleotide: the first RNA product and the 3'-end of the priming oligonucleotide is extended by DNA polymerase to produce a second DNA primer extension product complementary to the first RNA product . See FIG. 1, Stages 10-12. The second primer extension product is then separated from the first RNA product using an enzyme that selectively degrades the first RNA product (e.g., with RNAse H activity). See FIG. 1, Stage 13.

[0187] The second primer extension DNA product is treated with a promoter oligonucleotide that hybridizes to the 3'-end of the second primer extension DNA product to form a hybrid promoter oligonucleotide: second primer extension DNA product. See FIG. 1, Step 14. Hybrid promoter oligonucleotide: the second DNA primer extension product then re-enters the amplification cycle in Step 6 of FIG. The method of claim 1, wherein the transcription begins with a double-stranded promoter and the cycle is continued.

[0188] FIG. 3 illustrates the adaptation of an isothermal transcription-based amplification reaction called transcription-mediated amplification (TMA), various aspects of which are disclosed in Kacian et al., US Patent Nos. 5,399,491 and 5,824,518. The reaction from this exemplary embodiment begins by acting on the target sequence RNA in a nucleic acid sample labeled with a promoter oligonucleotide. The tagged promoter oligonucleotide comprises a tag sequence, a target hybridizing sequence, and a promoter sequence for RNA polymerase, wherein the target hybridizing sequence hybridizes to the 3'-end of the target sequence. Thus, the target sequence forms a stable complex with the tagged promoter oligonucleotide at the 3'-end of the target sequence before starting the primer extension reaction. See FIG. 3, Stage 1. The promoter sequence is located 5 'to the tag sequence and the tag sequence is located 5' to the target hybridizing sequence. The unhybridized tagged promoter oligonucleotide is made available for hybridization with the target sequence prior to starting the primer extension reaction with the tagged priming oligonucleotide, preferably by inactivating and / or removing the unhybridized tagged priming oligonucleotide from the nucleic acid sample.

[0189] Then, from the 3'-end of the tagged priming oligonucleotide, a DNA polymerase extension reaction, e.g., reverse transcriptase, begins to form the first DNA primer extension product that includes the tag and promoter sequences and a region complementary to the target sequence. See FIG. 1, Steps 2 and 3. The first primer extension product is then separated from the target sequence with which it is hybridized using an enzyme that selectively degrades that portion of the target sequence that is hybridized with the first DNA primer extension product (e.g., with RNAse H activity). See FIG. 3, Stage 4.

[0190] Next, the first primer extension DNA product is treated with a primer oligonucleotide that hybridizes to a region of the first primer extension DNA product that is complementary to the 5'-end of the target sequence, thereby forming the hybrid primer oligonucleotide: the first primer extension DNA product. See FIG. 3, Stage 5. The 3'-end of the priming oligonucleotide is extended by DNA polymerase to form a second DNA primer extension product complementary to at least a fragment of the first DNA primer extension product and containing a double-stranded promoter sequence. See FIG. 3, Steps 6 and 7. This second DNA primer extension product is used as a template for transcription of multiple copies of the first RNA product complementary to the second DNA primer extension product, not including the promoter portion, using RNA polymerase which recognizes the double-stranded promoter and begins transcription from it. See FIG. 3, Step 8 and 9. The base sequence of the first RNA product is substantially identical to the base sequence of the tag sequence and the complement of the target sequence.

[0191] The first RNA product is treated with a priming oligonucleotide whose 3 'end is extended by DNA polymerase to form a third DNA product that extends the primer complementary to the first RNA product. See FIG. 3, Stages 10-12. The third DNA primer extension product is then separated from the first RNA product using an enzyme that selectively degrades the first RNA product (e.g., with RNAse H activity). See FIG. 3, Stage 13. The third DNA primer extension product is treated with a primer oligonucleotide having a hybridizing sequence that hybridizes to the complement of the tag sequence at the 3'-end of the third DNA primer extension product, and further includes a RNA polymerase promoter that is located 5 'to the hybridizing sequence . See FIG. 3, Step 14. The 3'-end of the third DNA primer extension product is extended to attach a sequence complementary to the promoter sequence. See FIG. 3, Step 15. The 3'-end of the promoter oligonucleotide is extended by DNA polymerase to form a fourth DNA primer extension product complementary to a third DNA primer extension product. See FIG. 3, Step 16. Multiple copies of the second RNA product complementary to the third DNA primer extension product, not including the promoter fragment, are transcribed from the double-stranded promoter and re-enter the amplification cycle in Step 9 of FIG. 3. The base sequence of the second RNA product is substantially identical to the base sequence of the tag sequence and the complement of the target sequence.

[0192] FIG. 5 illustrates the adaptation of rTMA amplification reactions to amplification of a target DNA sequence, various aspects of which are disclosed in Becker et al., US Patent Application No. 11/681104. The reaction of this exemplary embodiment is initiated by treating the target DNA sequence in a nucleic acid sample labeled with a priming oligonucleotide and a terminating oligonucleotide. The tagged priming oligonucleotide comprises a target hybridizing sequence hybridized to the 3'-end of the target sequence and a tag sequence located 5 'to the target hybridizing sequence. The target hybridizing sequence preferably hybridizes to the single-stranded form of the target sequence, although it can hybridize to the double-stranded form of the target sequence by invading the strand, which can be facilitated, e.g., by loosening DNA (e.g. regions rich in AT), low salt conditions and / or the use of DMSO and / or osmolytes such as betaine. The target sequence is preferably carried out in single-stranded by heating the nucleic acid sample. The terminating oligonucleotide hybridizes to the target nucleic acid region containing the target sequence, near the 5'-end of the target sequence. The terminating oligonucleotide is used to terminate primer extension in a nascent nucleic acid that includes a tagged priming oligonucleotide. Thus, the target nucleic acid forms a stable complex with the tagged priming oligonucleotide at the 3'-end of the target sequence and with the terminating oligonucleotide located adjacent to or near the 5'-end of the target sequence. See FIG. 5, Stages 1-3. The unhybridized tagged priming oligonucleotide is made unavailable for hybridization with the target sequence before starting the primer extension reaction with the tagged priming oligonucleotide, preferably by inactivating and / or removing the unhybridized tagged priming oligonucleotide from the nucleic acid sample.

[0193] Then, from the 3'-end of the tagged priming oligonucleotide, a DNA polymerase extension reaction, e.g., reverse transcriptase, begins to form a first DNA primer extension product that includes a tag sequence and a region complementary to the target sequence. See FIG. 5, Steps 4 and 5.

[0194] The nucleic acid sample is then treated with a displacement oligonucleotide that hybridizes to the target nucleic acid upstream of the tagged oligonucleotide so that the primer extension reaction can be started therefrom, such that the first DNA primer extension product is displaced when The 3'-end of the displacement oligonucleotide is extended by DNA polymerase. See FIG. 5, Stages 6-8. The order of the illustrated steps is not intended to mean that the nucleic acid sample of this form should be treated with a labeled priming oligonucleotide prior to treatment with the displacement oligonucleotide to make it functional. In certain embodiments, the simultaneous hybridization of these two oligonucleotides to the target nucleic acid sequence is generally preferred.

[0195] Next, the first DNA primer extension product is treated with a promoter oligonucleotide having a hybridizing sequence and an RNA polymerase promoter located 5 'to the hybridizing sequence. The hybridizing sequence hybridizes to the region of the first DNA primer extension product that is complementary to the 3'-end of the target sequence, thereby forming a hybrid promoter oligonucleotide: the first DNA primer extension product. In the illustrated reaction, the promoter oligonucleotide is modified to prevent the initiation of DNA synthesis by placing a blocking moiety at the 3'-end of the promoter oligonucleotide (e.g., 3'-to-5 'orientation nucleotide sequence). See FIG. 5, Step 9. The 3'-end of the first DNA primer extension product is extended to attach sequences complementary to the promoter, resulting in the formation of a double-stranded promoter sequence. See FIG. 5, Steps 10 and 11. Numerous copies of the first RNA product complementary to at least a fragment of the first DNA primer extension product, not including the promoter sequence, are transcribed using RNA polymerase, which recognizes the double-stranded promoter and begins transcription from it. See FIG. 5, Step 12 and 13. As a result, the base sequence of the first RNA product is substantially identical to the base sequence of the target sequence and the complement of the tag sequence.

[0196] The first RNA products are contacted with a priming oligonucleotide that hybridizes to the complement of the tag sequence to form a hybrid primer oligonucleotide: the first RNA product, and the 3'-end of the priming oligonucleotide is extended by a DNA polymerase to produce a second DNA primer extension product complementary to the first product RNA. See FIG. 5, Stages 14-16. The second DNA primer extension product is separated from the first RNA product using an enzyme that selectively degrades the first RNA product (e.g., with RNAse H activity). See FIG. 5, Stage 17.

[0197] The second DNA primer extension product is treated with a promoter oligonucleotide to form a hybrid promoter oligonucleotide: second DNA primer extension product. See FIG. 5, Step 18. Hybrid promoter oligonucleotide: the second primer extension product then re-enters the amplification cycle in Step 10 of FIG. The method of claim 5, wherein transcription begins with the double-stranded promoter and the cycle continues.

[0198] FIG. 7 illustrates polymerase chain reaction (PCR) adaptation, various aspects of which are disclosed, for example, in Mullis et al., US Patent Nos. 4,683195 and 4,800,195; Mullis, U.S. Patent No. 4,682,202; and Gelfand et al., U.S. Patent No. 5,804375. The reaction of this exemplary embodiment is initiated by treating the denatured target DNA sequence in a nucleic acid sample labeled with a priming oligonucleotide. The tagged priming oligonucleotide comprises a target hybridizing sequence that hybridizes to the 3'-end of the target sequence and a tag sequence located 5 'to the target hybridizing sequence. Thus, the target sequence forms a stable complex with the tagged priming oligonucleotide at the 3'-end of the target sequence before starting the primer extension reaction. See FIG. 7, Stages 1-3. The unhybridized tagged priming oligonucleotide is made unavailable for hybridization with the target sequence prior to initiating the primer extension reaction with the priming oligonucleotide, preferably by inactivating and / or removing the tagged starter oligonucleotide from the nucleic acid sample.

[0199] Then, from the 3'-end of the tagged priming oligonucleotide, a DNA polymerase extension reaction, e.g., Taq DNA polymerase, begins to form a first primer extension product that includes a tag sequence and a region complementary to the target sequence. See FIG. 7, Steps 4 and 5. The double-stranded product resulting from the first primer extension reaction is then denatured and the first primer extension DNA product is contacted with the first primer oligonucleotide that hybridizes with the region of the first primer extension DNA product that is complementary to the 5'-end of the target sequence. See FIG. 7, Stages 6 and 7.

[0200] In the second primer extension reaction, the 3'-end of the first primer oligonucleotide is extended by DNA polymerase to form a second DNA primer extension product that is complementary to a fragment of the first primer extension product and includes the target sequence and the complement of the tag sequence. See FIG. 7, Stages 8 and 9. The double-stranded product resulting from the second primer extension reaction is denatured and the second primer extension DNA product is contacted with a second primer oligonucleotide that hybridizes to the complement of the tag sequence. See FIG. 7, Steps 10 and 11.

[0201] The 3 'end of the second primer oligonucleotide is then extended in a third primer extension reaction using DNA polymerase to form a third primer extension DNA product that is complementary to the second primer extension DNA product. FIG. 7, Stages 12 and 13. The double-stranded product resulting from the third primer extension reaction is denatured and the second and third DNA primer extension products are available to participate in repeated cycles of the polymerase chain reaction using the first and second primer oligonucleotides as primers. See FIG. 7, Stages 14-16.

[0202] FIG. 9 illustrates the adaptation of reverse transcription polymerase chain reaction (RT-PCR), various aspects of which are disclosed, for example, in Gelfand et al., US Patent Nos. 5,322,770 and 5,315,652. The reaction of this exemplary embodiment begins by treating the target RNA sequence in oligonucleotide tagged primer sample. The tagged priming oligonucleotide comprises a target hybridizing sequence and a tag sequence located 5 'to the target hybridizing sequence. Thus, the target sequence forms a stable complex with the tagged priming oligonucleotide at the 3'-end of the target sequence before starting the primer extension reaction. See FIG. 9, Stage 1. The unhybridized tagged priming oligonucleotide is made unavailable for hybridization with the target sequence prior to starting the primer extension reaction with the tagged priming oligonucleotide, preferably by inactivating and / or removing the unhybridized tagged priming oligonucleotide from the nucleic acid sample.

[0203] Then, from the 3'-end of the tagged priming oligonucleotide, a DNA polymerase extension reaction, e.g., Tth MasterAmp ™ DNA polymerase, begins to form a first DNA primer extension product that includes a tag sequence and a region complementary to the target sequence. See FIG. 9, Stages 2 and 3. The first DNA primer extension product is then separated from the target nucleic acid sequence with which it is hybridized using an enzyme that selectively degrades that fragment of the target nucleic acid containing the target sequence that is complementary to the first DNA primer extension product (e.g., RNAse H activity ). See FIG. 9, Stage 4.

[0204] Next, the first primer extension DNA product is treated with a first primer oligonucleotide that hybridizes to a region of the first primer extension DNA product that is complementary to the 5'-end of the target sequence, forming a hybrid first primer extension DNA product: the first primer oligonucleotide. See FIG. 9, Stage 5. In the second primer extension reaction, the 3'-end of the first priming oligonucleotide is extended by DNA polymerase to form a second DNA primer extension product complementary to at least a fragment of the first primer extension product and comprising the target sequence and the complement of the tag sequence. See FIG. 9, Steps 6 and 7. The first and second DNA primer extension products are then separated from each other by denaturation. See FIG. 9, Stage 8. The first and second primer extension products are then available to participate in repeated cycles of the polymerase chain reaction using as a primer the first priming oligonucleotide and the second priming oligonucleotide that hybridizes to the complement of the tag sequence. See FIG. 9, Steps 9 and 10; FIG. 7, Stages 13-16.

[0205] In other exemplary embodiments of the present disclosure, a heterologous tag sequence that has not become part of the tagged target nucleic acid sequence is inactivated prior to exposing the tagged target nucleic acid sequence to reagents and sufficient conditions for detectably amplifying the target nucleic acid sequence. In a preferred aspect, the inactivated heterologous tag sequence is in the form of a tagged oligonucleotide that has not hybridized to the target nucleic acid sequence. The tagged oligonucleotides are described above and include the first and second regions, wherein the first region comprises a target hybridizing sequence that hybridizes to the 3'-end of the target nucleic acid sequence under the first set of conditions, and the second region comprises a tag sequence located 5 'from the first region of the tagged oligonucleotide. The target hybridizing sequence has a free 3 'hydroxyl group that can be enzymatically extended in the presence of DNA polymerase in a template-dependent manner. The tagged oligonucleotide has an "active" conformation that allows the target hybridizing sequence to hybridize to the target nucleic acid sequence and a "inactive" conformation that blocks the hybridization of the target hybridizing sequence to the target nucleic acid sequence. The inactive conformation is generally created under less stringent conditions than the conditions for forming the active conformation of the tagged oligonucleotide.

[0206] The inactive conformation of the tagged oligonucleotide may be formed by hybridizing the tag closing sequence with the target hybridizing sequence from the tagged oligonucleotide. The tag closing sequence may be a separate molecule or may be linked to a tagged oligonucleotide via a linker that connects the 3'-end or 5'-end of the tag closing sequence to the 5'-end of the tagged region of the oligonucleotide containing the tag sequence ("tagged priming oligonucleotide") or a promoter sequence located 5 'to the tag sequence ("tagged promoter oligonucleotide"), thereby forming a tag molecule with a hairpin structure resulting from auto-hybridization, including the tagged oligonucleotide. The linker contains no nucleotide bases that can be amplified by a polymerase and is preferably a non-nucleotide linker composed of non-nucleotide elements. Suitable non-nucleotide linkers for linking the tag closing sequence to the tagged oligonucleotide include baseless nucleotides and polyethylene glycol. Other suitable linkers include nucleotide analogs such as LNA and 2'-O-Me. Binding kinetics are best when the tag closing sequence and the target hybridizing sequence from the tagged oligonucleotide are on the same molecule.

[0207] Under selective conditions, the tag closing sequence can hybridize to the target hybridizing sequence from the tagged oligonucleotide in anti-parallel orthosis as shown in Figures 2, 4, 6, 8, 10, 11, 12, 15 and 16, or in orientation parallel as shown in Figures 13 and 14. If the tag closing sequence is a separate molecule as illustrated in Figures 11 and 12, or is connected to the tagged oligonucleotide via a non-nucleotide linker linked to its 5'-terminus as illustrated in Figures 2, 4, 6, 8, 10, 15 and 16 , then the tag closing sequence is preferably modified to prevent primer extension by DNA polymerase, for example by placing a blocking moiety at its 3'-end. Suitable blocking moieties are described herein. When hybridized in an anti-parallel orientation as illustrated in Figures 13 and 14, the 3'-terminal base of the tag closing sequence is preferably hybridized to the 3'-terminal base of the hybridizing sequence with the target from the tagged oligonucleotide. More preferably the tag closing sequence is modified to prevent primer extension by DNA polymerase.

[0208] The length and content of bases in the tag closing sequence are selected so that hybridization of the tagged oligonucleotide with the target nucleic acid sequence is favored in the first set of conditions, and so that the tag closing sequence can form a stable hybrid with the target hybridizing sequence in the second, a less stringent set of conditions when the tagged oligonucleotide is not hybridized to the target nucleic acid sequence. The tag closing sequence should be chosen so that it is not readily displaced from the target hybridizing sequence under amplification conditions to which it may be exposed. Typically, the tag closing sequence will hybridize to from 5 to 20 adjacent or non-contiguous bases of the target hybridizing sequence. Suitable tag closing sequences are preferably in the range of 5 to 15 bases in length. In order to favor the hybridizing sequence with the target nucleic acid target in the first set of conditions, the tag closing sequence may comprise, for example, one or more non-base nucleotides, base mismatches or elements of shaky base pairs. The tag closing sequences are preferably selected so that they specifically hybridize to the target hybridizing sequence more strongly than all non-specific interactions with other nucleic acids present in the amplification reaction.

[0209] After inactivation, inactive tagged oligonucleotides are preferably removed from the sample to limit unintended interactions with target nucleic acid sequences delivered to the sample from a potential contaminating source. Removal can be achieved by immobilizing target nucleic acids in the sample on a solid support, followed by removal of other components from the sample, including inactivated tagged oligonucleotides. To ensure that inactive labeled oligonucleotides are removed, the number of non-specific interactions between the solid support and nucleic acids present in the sample should be limited. Any solid support for the sample can be used, such as matrices and particles that are free in solution. Particularly preferred solid substrates are magnetic balls that are homogeneous (i.e. of equal size (5%), thus ensuring consistent results, which is particularly advantageous for use in an automated procedure.

[0210] Particularly preferred amplification techniques for incorporating the tagged oligonucleotides of the present invention include isothermal amplification reactions, such as TMA and TMA variants, such as real-time TMA, which include one or more features of the methods described by Becker et al., US Patent Application Publication No. US 2006-0046265 A1, and Becker et al., US Patent Application No. 11 / 681,104. For example, certain preferred real-time TMA methods include the use of blocking moieties, terminating moieties, and / or other modifying moieties that provide improved sensitivity and accuracy of the TMA process.

[0211] Promoter oligonucleotides may be modified to prevent DNA synthesis from them. For example, the promoter oligonucleotide may contain a blocking moiety attached to its 3'-terminus to prevent primer extension in the presence of polymerase. In one example, at least about 80% of the oligonucleotides present in the amplification reaction that contain the promoter further include a 3'-blocking moiety. In another embodiment, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the oligonucleotides provided for the amplification reaction that contain the promoter is further modified to contain a 3'-moiety blocking. In another embodiment, each oligonucleotide used in the amplification reaction of the present disclosure that contains a promoter sequence further includes a 3'-blocking moiety. [0212] Specific embodiments of the present disclosure relate to the amplification of a target nucleic acid comprising a target RNA sequence. In some cases, the target nucleic acid has unspecified 3 'and 5' ends relative to the desired target RNA sequence. The target nucleic acid is treated with a priming oligonucleotide that has a base region sufficiently complementary to the 3'-end of the target RNA sequence to hybridize with it, and as discussed above, further includes a heterologous tag sequence in the first primer extension reaction. Primer oligonucleotides are designed to hybridize to the appropriate region of each desired target sequence in accordance with primer design methods well known to those of ordinary skill in the art. While the presence of a tag sequence in a priming oligonucleotide can change the binding properties of a target hybridizing region to a target nucleic acid sequence, one skilled in the art of molecular biology can easily design primer oligonucleotides that contain both target hybridizing regions and tag sequences that can be used as described herein ways. Suitable priming oligonucleotides are described in more detail herein. Additionally, the 5'-end of the priming oligonucleotide (preferably, the unlabelled priming oligonucleotide) may include one or more modifications that improve binding properties (e.g. hybridization or base setting) of the priming oligonucleotide with the DNA extension product or RNA amplification product, as discussed in more detail above, provided that these modifications do not significantly interfere with the primer oligonucleotide primer function or the cleavage of the RNA amplification product with which the priming oligonucleotide is hybridized. The 3'-end of the priming oligonucleotide is extended by a suitable DNA polymerase, e.g. RNA-dependent DNA polymerase ("reverse transcriptase") in the extension reaction using the target RNA sequence or amplification product as template, resulting in a DNA primer extension product that is complementary to the RNA template or amplification product.

[0213] The DNA primer extension products are separated (at least in part) from the RNA template using an enzyme that degrades the RNA template or amplification product. Suitable enzymes, ie, "selective RNAse," are those that act on the RNA: RNA complex DNA strand and include enzymes that have RNAse H activity. Some reverse transcriptases have RNAse H activity, including those derived from the Moloney murine leukemia virus. and avian myeloblastosis virus. According to preferred forms of amplification, selective RNAse may be provided as reverse transcriptase RNAse H activity or may be provided as a separate enzyme, e.g. as E. coli RNAse H or T. thermophilus RNAza H. Other enzymes that selectively degrade RNA present in the RNA: DNA duplex can also be used.

[0214] When the target sequence is DNA, the DNA primer extension product can be separated from the template by treatment of the target nucleic acid with a displacement oligonucleotide. The displacement oligonucleotide has a primer function and is designed to hybridize with the target nucleic acid upstream of the priming oligonucleotide (referred to in this form as the "forward priming oligonucleotide"). By "up" is meant that the 3'-end of the displacer oligonucleotide hybridizes to the target nucleic acid upstream of the 3'-end of the forward primer oligonucleotide. Thus, the displacer oligonucleotide and forward primer oligonucleotide can hybridize to overlapping or distinct regions of the target nucleic acid. In preferred embodiments, the 3'-end of the displacement oligonucleotide adheres to or is separated by 5 to 35 bases from the 5'-end of the oligonucleotide forward primer relative to the target nucleic acid (i.e., the target nucleic acid has up to 5 to 35 consecutive unbound nucleotides located between The 3'-base of the displacement oligonucleotide and the 5'-base of the priming oligonucleotide when both oligonucleotides are hybridized to the target nucleic acid). The displacement oligonucleotide is typically 10 to 50 nucleotides in length and may contain one or more 5'-end modifications that improve the binding properties (e.g., hybridization or base stacking) of the displacement oligonucleotide with the target nucleic acid, provided that these modifications do not interfere indeed, the displacement oligonucleotide primer function. The displacer oligonucleotide and forward primer oligonucleotide are designed to hybridize to the target nucleic acid under the same conditions. Preferably, the target nucleic acid is treated with the displacement oligonucleotide after the forward primer oligonucleotide has already had sufficient time to hybridize with the target nucleic acid. Alternatively, the target nucleic acid is treated with both the displacement oligonucleotide and the forward primer oligonucleotide prior to exposing the mixture to a polymerase suitable for extending the 3'-ends of the displacement oligonucleotide and the forward priming oligonucleotide. In the presence of DNA polymerase, the 3'-end of the displacement oligonucleotide is extended in a matrix-dependent manner to produce a second primer extension DNA product that displaces the first primer extension DNA product from the target nucleic acid, thus making it available for hybridization with the promoter oligonucleotide. In an alternative approach, conditions can be established in which the promoter oligonucleotide accesses the first DNA primer extension product by invading the strand facilitated by, for example, DNA relaxation (e.g. in AT rich regions), low salt conditions and / or use DMSO and / or osmolytes such as betaine. The promoter oligonucleotide of this form is the same as that described above and is likewise modified to prevent the promoter oligonucleotide from acting as a DNA polymerase priming oligonucleotide (e.g., the promoter oligonucleotide has a blocking moiety at its 3'-end).

[0215] In certain embodiments, the methods of the present disclosure further include treating the target nucleic acid as described above to limit the length of the DNA primer extension product to a specific desired length. This length limitation is usually achieved by using a "binding molecule" that hybridizes or otherwise binds to the target RNA nucleic acid adjacent to or near the 5'-end of the desired target sequence. In certain embodiments, the binding molecule comprises a base region. The base region may be DNA, RNA, a DNA: RNA chimeric molecule or an analogue thereof. Binding molecules containing a base region can be modified in one or more ways as described elsewhere herein. Suitable binding molecules include, but are not limited to, a binding molecule comprising a terminating oligonucleotide or terminating protein that binds to RNA and prevents primer from extending beyond its binding region, or a binding molecule comprising a modifying molecule, for example a modifying oligonucleotide such as a "digesting" oligonucleotide directs the hydrolysis of this fragment of the target RNA that is hybridized to a digestion oligonucleotide or sequence specific nuclease, which crosses the target RNA.

[0216] Exemplary terminating oligonucleotides of the present disclosure have a 5'-base region sufficiently complementary to the target nucleic acid in a region adjacent to or located in the vicinity of, or coinciding with the 5'-end of the target sequence to hybridize with it. In certain embodiments, the terminating oligonucleotide is synthesized to contain one or more modified nucleotides. For example, specific terminating oligonucleotides contain one or more 2'-O-ME-ribonucleotides or are entirely synthesized from 2'-O-ME-ribonucleotides. See, e.g., Majlessi et al. (1998) Nucleic Acids Res., 26, 2224-2229. The terminating oligonucleotide also typically has a blocking moiety at its 3'-terminus to prevent the terminating oligonucleotide from acting as a DNA polymerase primer. In certain embodiments, the 5'-end of the terminating oligonucleotide coincides and is complementary to at least about 2 nucleotides from the 5'-end of the target sequence. Typically, the 5'end of the terminating oligonucleotide of the present invention coincides with and is complementary to at least 3, 4, 5, 6, 7 or 8 nucleotides from the 5'-end of the target sequence, but no more than 10 nucleotides from the 5'-end of the target sequence . (As used herein, the term "end" refers to the 5'- or 3'-region of an oligonucleotide, nucleic acid or nucleic acid region, which includes the 5 'or 3'-base of an oligonucleotide, nucleic acid or nucleic acid region, respectively. ) Suitable terminating oligonucleotides are described in more detail herein.

[0217] A single-stranded primer extension DNA product or "first" primer extension DNA product that has either a 3'-defined or undefined 3'-end is treated with a promoter oligonucleotide that includes a first region sufficiently complementary to the 3'-region of the DNA product extending the primer to hybridize to it, a second region containing the RNA polymerase promoter, e.g., T7 polymerase, which is located 5 'to the first region, e.g. directly to the 5 'side or separated from the first region and modified to prevent the promoter oligonucleotide from acting as a DNA polymerase primer (e.g., the promoter oligonucleotide contains a blocking moiety associated with its 3'-terminus). Once the desired hybridizing "first region" has been identified, convenient promoter oligonucleotides can be constructed by one of ordinary skill in the art using only routine procedures. Those of ordinary skill in the art will readily understand that the promoter region has specific nucleotides that are required for recognition by a given RNA polymerase. In addition, specific nucleotide changes in the promoter sequence can improve promoter interaction with a given enzyme, including the use of inserted sequences.

[0218] Insertion sequences can be inserted between the first and second regions of promoter oligonucleotides and act by increasing the rate of amplification. (The tag sequence of the tagged promoter oligonucleotide may provide this beneficial effect.) Improved amplification rates may be associated with various factors. First, because the inserted sequence increases the distance between the 3'-end and the promoter sequence from the promoter oligonucleotide, there is less likelihood that the polymerase, e.g. reverse transcriptase, associated with the 3'-end of the promoter oligonucleotide will interfere with binding of RNA polymerase to the promoter sequence, thereby increasing same speed at which transcription can be started. Secondly, the selected inserted sequence can itself improve the transcription rate by acting as a better transcription matrix than the target sequence. Third, as the RNA polymerase will start transcription from the inserted sequence, the primer extension product synthesized by the priming oligonucleotide, using the RNA transcription product as template, will contain the complement of the sequence inserted towards the 3'-end of the primer extension product. By providing a larger target binding region, i.e., one that includes completion of the insertion sequence, the promoter oligonucleotide can bind to the primer extension product more quickly, thereby leading to the production of additional RNA transcription products earlier. The inserted sequences are preferably 5 to 20 nucleotides in length and should be designed to minimize intramolecular folding and intermolecular binding to other oligonucleotides present in the amplification reaction mixture. Programs that help minimize secondary structure are well known in the art and include Michael Zucker mfold software for predicting RNA and DNA secondary structure using thermodynamic principles in the immediate vicinity. The latest version of Michael Zucker's mfold software can be obtained online at<a href="http://www.bioinfo.rpi.edu/cipplicationstmfold">www.bioinfo.rpi.edu/cipplicationstmfold</a> using a protocol for sending hypertext documents (http) for a URL. Useful inserted sequences can be identified using in vitro selection methods well known in the art without involving anything more than routine experimentation.

[0219] Assessment of promoter oligonucleotides with differences in promoter sequences can be easily carried out by one skilled in the art using routine methods. In addition, if the use of another RNA polymerase is desired, the promoter sequence in the promoter oligonucleotide may be easily substituted by another promoter. Substitution of other promoter sequences is within the knowledge and skills of those of ordinary skill in the art. In real-time TMA, the promoter oligonucleotides supplied to the amplification reaction mixture are modified to prevent the successful start of DNA synthesis from their 3'-ends, and preferably include a blocking moiety associated with their 3'-ends. In addition, oligonucleotide end cleotides and cap oligonucleotides, and even probes used in certain embodiments also optionally include a blocking moiety associated with their 3 'ends.

[0220] When a terminating oligonucleotide is used, the first priming oligonucleotide region is designed to hybridize to the desired 3'-end of the DNA primer extension product with substantial, but not necessarily perfect accuracy. The second promoter oligonucleotide region may then act as a template, allowing further extension of the first DNA primer extension product to attach a base region complementary to the second promoter oligonucleotide region, i.e. region containing the promoter sequence to form a double-stranded promoter. The RNA polymerase that recognizes the promoter binds to the promoter sequence and begins to transcribe multiple copies of RNA complementary to the DNA primer extension product, which copies are substantially identical to the target sequence. By "substantially identical" it is meant that multiple copies of RNA may have additional nucleotides, either on the 5 'or 3' side of the target sequence or may have fewer nucleotides, either on the 5 'or 3' side of the target sequence, depending on e.g., "target sequence" boundaries, transcription initiation site, or whether the priming oligonucleotide has additional 5 'nucleotides from the primer region (e.g., a bound "cap" as described herein). When the target sequence is DNA, the RNA copy sequence is described herein as "substantially identical" to the target sequence. It should be understood, however, that the RNA sequence that contains uridine residues at the thymidine residue site of the target DNA sequence still has a "substantially identical" sequence. Thus produced RNA transcripts can automatically re-cycle from the above system without further manipulation. So the reaction is autocatalytic. In those embodiments in which the binding molecule or other methods of terminating the primer extension reaction are not used , the first primer oligonucleotide region is designed to hybridize with the selected region of the first DNA primer extension product that is expected to be 5 'to 3' - end of the first DNA primer extension product, but because the 3'-end of the first DNA primer extension product is indefinite, the region, in which the promoter oligonucleotide hybridizes is unlikely to be the actual 3'-end of the first DNA primer extension product. According to this embodiment, typically the at least 3'-base of the first DNA primer extension product does not hybridize to the promoter oligonucleotide. Thus, according to this embodiment, the first DNA primer extension product will probably not be further extended to form a double-stranded promoter.

[0221] The formation of a double-stranded promoter sequence by extending the template nucleic acid is not necessary to allow the start of RNA transcription complementary to the first DNA primer extension product. The resulting "first" RNA products are substantially identical to the target sequence and have a 5'-end defined by the transcription start point and a 3'-end defined by the 5'-end of the first DNA primer extension product. A sufficient number of the first RNA products are produced to automatically re-enter the system cycles without further manipulation. The priming oligonucleotide hybridizes to the 3'-end of the first RNA products and is extended by DNA polymerase to form a second DNA primer extension product. Unlike the first DNA primer extension product formed without the use of a terminating oligonucleotide or other binding molecule, the second DNA primer extension product has a defined 3'-end which is complementary to the 5'-ends of the first RNA products. The second DNA primer extension product is separated (at least in part) from the RNA template using an enzyme that selectively degrades the RNA template. The single-stranded second DNA primer extension product is then treated with a promoter oligonucleotide as described above, and the second promoter oligonucleotide region acts as a template, allowing further extension of the second DNA primer extension product to attach a base region complementary to the second promoter oligonucleotide region, i.e., the containing region promoter sequence to form a double-stranded promoter. The RNA polymerase that recognizes the promoter binds to the promoter sequence and begins to transcribe numerous "second" RNA products complementary to the second DNA primer extension product and substantially identical to the target sequence. The second RNA transcripts so produced automatically re-enter cycles from the above system without further manipulation. So the reaction is autocatalytic.

[0222] In any of the embodiments described above, after identifying the desired region in the target sequence, this region can be analyzed to determine sites where selective RNAse degradation will result in optimal cleavage or removal of RNA sections from the RNA: DNA duplex. Analyzes may be performed to evaluate the effect of RNAse degradation of the target sequence due to AMV RNAse H reverse transcriptase or MMLV reverse transcriptase activity, by exogenously added selective RNAse enzyme, e.g. RNAse H from E. coli or selective enzymes with RNAse activity from other sources and their combinations. After such analyzes, the priming oligonucleotide can be selected to hybridize to a segment of RNA that is substantially not degraded by the selective RNAase present in the reaction mixture, since substantial degradation at the binding site for the priming oligonucleotide could inhibit the start of DNA synthesis and prevent optimal primer extension. In other words, the priming oligonucleotide is typically selected to hybridize to the target RNA nucleic acid region or complement the target DNA nucleic acid, such that when the RNA is degraded by selective RNAse, there is essentially no degradation that would prevent formation of the primer extension product. [0223] In contrast, the hybridization site of the priming oligonucleotide can be selected such that sufficient degradation of the RNA strand occurs to allow efficient hybridization of the promoter oligonucleotide with the DNA strand. Typically, only RNA fragments are removed from the RNA: DNA duplex by selective RNAse degradation, and therefore some parts of the RNA strand will remain in the duplex. As a result of the degradation of RNA strands from the RNA: DNA hybrid by selective RNAse, small parts of RNA are separated from the hybrid. Positions at which RNA is selectively degraded can be determined by standard hybridization analysis. Therefore, one may choose a promoter oligonucleotide that will more effectively bind to DNA after selective degradation by RNAse, i.e. it will bind in areas from which RNA fragments are selectively removed.

[0224] Promoters or promoter sequences convenient for inclusion in the promoter oligonucleotides used in the methods of the present invention are nucleic acid sequences (naturally occurring, synthetically produced, or restriction digests) that are specifically recognized by RNA polymerase that recognizes and binds to this sequence and starts the transcription process in which RNA transcripts are produced. Typical known and useful promoters include those that are recognized by specific bacteriophage polymerases, such as those from T3, T7 and SP6 bacteriophages, and the E. coli promoter. The sequence may optionally include nucleotide bases that go beyond the actual recognition site of RNA polymerase, which may confer additional stability or susceptibility to degradation processes or increased transcription efficiency. Promoter sequences for which there is a known and available polymerase that is capable of recognizing the starting sequence are particularly convenient for use.

[0225] Suitable DNA polymerases for use in accordance with the methods of the disclosure include reverse transcriptases. Particularly convenient DNA polymerases include AMV reverse transcriptase and MMLV reverse transcriptase. Some of the reverse transcriptases suitable for use in the methods of the present invention, such as AMV and MMLV reverse transcriptases, have RNAse H activity. In fact, according to certain embodiments of the present invention, the only selective activity

RNAse H in the amplification reaction is provided by reverse transcriptase - no additional selective RNAse is added. However, in some situations it may also be useful to add exogenous selective RNAse, such as RNAse H from E. coli. Although the addition of exogenous selective RNAse is not required, under certain conditions RNAse H activity present in e.g. AMV reverse transcriptase may be inhibited or inactivated by other components present in the reaction mixture. In such situations, the addition of exogenous selective RNAse may be desirable. For example, when relatively large amounts of heterologous DNA are present in the reaction mixture, the natural RNAse H reverse transcriptase activity may be somehow inhibited, and thus the number of copies of the target sequence generated is accordingly reduced. In situations where the target nucleic acid includes only a small fragment of the current nucleic acid (e.g. when the sample contains significant amounts of heterologous DNA and / or RNA), it is particularly useful to add exogenous selective RNAse. See, e.g., Kacian et al., US Patent No. 5,399,491.

[0226] RNA amplification products produced by the methods described above can serve as templates for producing additional amplification products associated with the target sequence by the mechanisms described above. The system is autocatalytic and amplification by the methods of the present invention occurs without the need for repeated modification or alteration of reaction conditions such as temperature, pH, ionic strength and the like. These methods do not require expensive thermal cycle apparatus, nor do they require many steps to add enzymes or other reagents during the amplification reaction.

[0227] As emphasized above, the methods of the present disclosure are useful in assays for detecting and / or quantifying specific target nucleic acid sequences in clinical, water, environmental, industrial, beverage, food, seed and other samples, or for producing large quantities of RNA amplification products from a specific target sequence for a variety of applications. For example, the present invention is useful for screening clinical samples (e.g. blood, urine, feces, saliva, semen or spinal fluid), food, water, laboratory and / or industrial samples for the presence of specific nucleic acids, specific organisms (e.g. using species-specific oligonucleotides) and / or specific classes of organisms in applications such as sterility testing (e.g., using universal oligonucleotides). The present invention can be used to detect the presence of, for example, viruses, bacteria, fungi or parasites.

[0228] The amplification product can be detected by any standard methods. For example, the amplification product can be detected by hybridizing with a detectably labeled probe and measuring the resulting hybrids. Design criteria for selecting probes for detecting specific target sequences are well known in the art and are described, for example, in Hogan et al., "Methods for Making Oligonucleotide Probes for the Detection and / or Quantitation of Non-Viral Organisms," US Patent No. 6150517. Hogan states that probes should be designed to maximize homology with the target sequence (s) and minimize homology with possible non-target sequences. In order to minimize stability with non-target sequences, Hogan points out that guanine and cytosine-rich regions should be avoided, that the probe should include as many destabilizing mismatches as possible, and that the length of ideal complementarity to the non-target sequence should be minimized. On the contrary, the stability of the probe with the target sequence (s) should be maximized, regions rich in adenine and thymine should be avoided, probe hybrids: the target preferably ends with base pairs of guanine and cytosine, significant autocomplementarity should be generally avoided, and the melting point hybrid probe: the target should be about 2-10 ° C higher than the temperature in the test.

[0229] In a particular embodiment, the amplification product can be tested in a hybridization protective assay ("HPA") that involves hybridization of an oligonucleotide chemiluminescent probe with the target sequence, e.g., an acridine ester labeled probe ("AE"), selective hydrolysis of the chemiluminescent enzyme present in the non-sonilidium present and measuring the chemiluminescence produced by the remaining probes in the luminometer. See e.g. Arnold et al., "Homogenous Protection Assay," US Patent No. 5,283,174 and NORMAN C. NELSON et al., NONISOTOPIC PROBING. BLOTTING, AND SEQUENCING, ch. 17 (Larry J. Kricka ed., 2nd ed. 1995).

[0230] In further embodiments, the present disclosure provides quantitative real-time evaluation of the amplification process by the methods described herein. Assessment of the "real-time" amplification process involves determining the amount of amplicon in the reaction mixture continuously or periodically during the amplification reaction, and the specified values ​​are used to calculate the amount of target sequence initially present in the sample. There are many different methods for determining the amount of target sequence initially present in a sample based on real-time amplification. These include those disclosed by Wittwer et al., "Method for Quantification of an Analyte", US Patent No. 6,303,305, and Yokoyama et al., "Method for Assaying Nucleic Acid", US Patent No. 6,541,205. Another method for determining the amount of target sequence initially present in a sample but which is not based on real-time amplification is disclosed by Ryder et al., "Method for Determining Pre-Amplification Levels of a Nucleic Acid Target Sequence from Post-Amplification Levels of Product ", US Patent No. 5,710,029. Amplification products can be detected in real time by using various self-hybridizing probes, most of which have a" trunk and loop "structure. Such self-hybridizing probes are labeled to emit signals detectable in a variety of ways, depending on whether the probes are in a state resulting from auto-hybridization or in a state changed by hybridization with the target sequence. As an example, "molecular torches" probes are a type of self-hybridizing probe that includes separate regions of auto-complementarity (called "target binding domain" and "target closing domain") that are connected via a linking region (e.g. non-nucleotide linker) and which hybridize to each other under predetermined hybridization test conditions. In a preferred embodiment, the molecular torches probes contain single-stranded base regions in the target binding domain that are from 1 to about 20 bases in length and are available to hybridize to the target sequence present in the amplification product under strand displacement. Under thread displacement conditions, hybridization of two complementary regions (which may be completely or partially complementary) of the molecular torch probe is favored, unless a target sequence is present that binds to a single-stranded region present in the target binding domain and displaces all or part of the target closing domain. The target binding domain and target closing domain of the molecular torch probe include a detectable tag or a pair of interacting tags (e.g., luminescent compound / silencer) positioned so that a different signal is generated when the molecular torch probe is self-hybridized than when the probe type molecular torch is hybridized to the target sequence, thus enabling detection of probe: target duplexes in the sample under study in the presence of unhybridized molecular torch probes. Molecular torch probes and various types of interacting tag pairs are disclosed by Becker et al., "Molecular Touches," US Patent No. 6,534,274.

[0231] Another example of a self-complementary detection probe is the "molecular beacon" probe. Molecular beacon probes include nucleic acid molecules that complete the target sequence, an affinity pair (or nucleic acid arms) holding the probe in a closed conformation in the absence of the target sequence in the amplification product, and a pair of tags that interact with each other when the probe is in the closed conformation. Hybridization of the target sequence and completion of the target sequence results in the separation of affinity pair elements, thereby transforming the probe into an open conformation. A change to the open conformation is detectable due to the reduced interaction of the marker pair, which may be, for example, a fluorophore and a quencher (e.g. DABCYL and EDANS). Molecular beacon probes are disclosed by Tyagi et al., "Detectably Labeled Dual Confirmation Oligonucleotide Probes, Assays and Kits," US Patent No. 5925517, and Tyagi et al., "Nucleic Acid Detection Probes Having Non-FRET Fluorescence Quenching and Kits and Assays Including Such Probes ", US Patent No. 6,150097.

[0232] Other self-hybridizing probes are well known to those of ordinary skill in the art. As an example, binding pairs in probes having interacting labels, such as those disclosed by Morrison, "Competitive Homogenous Assay", US Patent No. 5928862, and Gelfand et al., US Patent No. 5,804375 for PCR reactions, can be adapted for use in this disclosure. Additional detection systems include "molecular switches" as disclosed by Arnold et al., "Oligonucleotides Comprising a Molecular Switch", US Patent Application Publication No. US 20050042638 A1. Also other probes, such as those containing intercalating dyes and / or fluorochromes, may be useful for detecting amplification products in the present disclosure. See, e.g., Ishiguro et al., "Method of Detecting Specific Nucleic Acid Sequences," US Patent No. 5,814,477.

[0233] In those methods where the initial target sequence and RNA transcription product have the same meaning, it may be desirable to start amplification before adding the probe for real-time detection. Addition of the probe before starting the amplification reaction may reduce the amplification rate as the probe that binds to the initial target sequence must be displaced or otherwise removed during the primer extension step to complete the primer extension product containing the complement of the target sequence. Start of amplification is defined as the addition of enzymes for amplification (e.g. reverse transcriptase and RNA polymerase).

[0234] In addition to the methods described herein, the present disclosure relates to kits comprising one or more reagents required to carry out the methods of the present disclosure. Kits comprising the various components used in carrying out the present disclosure can be selected for use in any procedure requiring the amplification of target nucleic acid molecules, and such kits can be adapted to a wide variety of end users. Convenient kits may be prepared, for example, for microbiological analysis, blood screening, disease diagnosis, water testing, product release or sterility testing, environmental or industrial analysis, food or beverage testing, or for general laboratory use. Kits provide one or more components necessary to perform nucleic acid amplification in accordance with the invention. Kits may include reagents convenient for amplifying nucleic acids from one specific target, or may include reagents convenient for amplifying multiple targets. The kits may further provide reagents for the real-time detection of one or more target nucleic acids in a single sample, for example one or more self-hybridizing probes, as described above. Kits may include a carrier which may be separated in separate compartments to obtain one or more containers, such as vials, test tubes, wells and the like. Preferably, at least one of such containers contains one or more ingredients or a mixture of ingredients needed to carry out the amplification methods of the present invention.

[0235] A kit according to one embodiment of the present disclosure may comprise, for example, in one or more containers a tagged oligonucleotide, alone or in combination with a tag closing oligonucleotide or linked to a tag closing sequence, a binding molecule, or other means to complete the primer extension reaction and optionally an elongating oligonucleotide and / or a cap oligonucleotide. If real-time detection is used, one or more containers may include one or more real-time detection reagents of at least one target nucleic acid sequence in a single sample, for example one or more self-hybridizing probes, as described above. Another container may contain an enzyme reagent, such as a thermostable DNA polymerase for performing a PCR or RT-PCR reaction, or a mixture of reverse transcriptase (with or without RNAse H activity), RNA polymerase, and optionally an additional selective RNAse H enzyme for transcription-based amplification reaction. These enzymes can be provided in concentrated form or in working concentration, usually in a form that increases the stability of the enzyme. The enzyme reagent may also be provided in lyophilized form. See Shen et al., "Stabilized Enzyme Compositions for Nucleic Acid Amplification", US Patent No. 5,834,254. Other of one or more containers may contain the amplification reagent in concentrated form, e.g., 10X, 50X, or 100X or at working concentration . The amplification reagent will contain one or more components necessary for carrying out the amplification reaction, e.g. buffer, MgCl2, KCl, dNTP, rNTP, EDTA, stabilizing agents, etc. Determined from the components, e.g. MgCl2 and rNTP, can be supplied separately from the other components, allowing the end user to measure these reagents for more optimized amplification reactions. One or more other containers may contain reagents for detecting amplification products, including one or more labeled oligonucleotide probes. Probes can be labeled with alternative methods, e.g., radioactive isotopes, fluorescent labels, chemiluminescent labels, nuclear labels, bioluminescent labels, intercalating dyes or enzyme labels. In some embodiments, the kit will also contain one or more containers containing one or more positive and negative control target nucleic acids that can be used in amplification experiments to confirm the amplifications tested by the end user. In some cases, one or more of the reagents listed above may be combined with internal control. Of course, it is also possible to combine one or more of these reagents in a single tube or other container. Substrates convenient for use with this disclosure, e.g., test tubes, multi-tube units, multi-well plates, etc. may also be provided in kits. Finally, the kit may include one or more instructions. [0236] The kits may contain potentially any combination of the above-described or elsewhere described ingredients. One of ordinary skill in the art will understand that the ingredients provided in the kits of the invention will vary depending upon the intended use of the kits and the intended end user. Thus kits may be specifically designed to perform the various functions described in this application and the components of such kits will change accordingly. [0237] The present disclosure further relates to various oligonucleotides, including, for example, specific target oligonucleotides as exemplified below. It should also be understood that the oligonucleotides of the present disclosure may be DNA, RNA, DNA chimeras: RNA and their analogs, and in each case, they include RNA oligonucleotide DNA equivalents and RNA oligonucleotide DNA equivalents.

[0238] Detection probes may include, for example, an acridine ester tag or labeled, self-hybridizing regions flanking a sequence that hybridizes to the target sequence. In various embodiments, these tagged oligonucleotide probes are optionally or preferably synthesized to contain at least one modified nucleotide, e.g. The 2'-O-ME-ribonucleotide or these labeled oligonucleotide probes are optionally or preferably synthesized entirely from modified nucleotides, e.g. 2'-O-ME-ribonucleotides.

EXAMPLES [0239] The following are examples illustrating specific aspects and embodiments of the invention.

[0240] Unless otherwise indicated, the oligonucleotides and modified oligonucleotides in the examples below were synthesized using standard phosphoramidite chemistry for which various methods are well known in the art. See, e.g., Carruthers et al., (1987) Meth .. Enzymol. 154, 287. Unless otherwise stated here, the modified nucleotides were 2'-O-ME-ribonucleotides, which were used in the synthesis as their phosphoramidite analogs.

EXAMPLE 1

SELECTIVE HCV AMPLIFICATION USING LABELED OLIGONUCLEOTIDES IN THE TMA REACTION IN REAL TIME [0241] The following series of experiments was performed to assess whether the use of a labeled oligonucleotide to modify the target nucleic acid sequence of the nucleic acid sample of the nucleic acid of the nucleic acid of the nucleic acid of the nucleic acid nucleic acid provided in the nucleic acid sample of interest, without simultaneously amplifying the target nucleic acid sequence provided from sources other than the nucleic acid sample of interest.

[0242] The reagents and conditions of the protocol used in the experiments carried out, as well as a discussion of the results and conclusions of the experiments, are presented below.

I. OLIGONUCLEOTIDES [0243] Unless otherwise indicated, oligonucleotides were synthesized using Expedite ™ 8909 DNA Synthesizer (PerSeptive Biosystems, Framingham, MA) using standard phosphite phosphite chemistry. See, e.g., Carruthers et al. (1987) Meth. Enzymol. 154, 287. Se25 sequences are in a 5-to-3 'orientation. The blocking moiety, when present, is at the 3'-end.

1. Tagged priming oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACAGGCATTGAGCGGGTTGATCCA AGAAAGGAC (SEQ ID NO: 1); 12 pmol / reaction mixture

2. Primer oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 2); 12 pmol / reaction mixture

3. Promoter oligonucleotide:

ATTTAATACGACTCACTATAGGGAGACCACAACGGTTTCTAGCCATGGCG TTAGTATGAG (SEQ ID NO: 3); 12 pmol / reaction mixture

Blocking moiety: 3'-to-3 'linker generated using 3'-dimethyltrityl84

N-benzoyl-2'-deoxycytidine, 5'-succinyl- (long chain alkyl) -amino-CPG (Glen Research Corporation, Sterling, VA; cat. No. 20-0102-01)

4. The terminating oligonucleotide:

AmUmGmGmCmUmAmGmAmCmGmCmUmUmUmCmUmGmCniGinDmGmA mAmGmAm (SEQ ID NO: 4); 0.8 pmol / reaction mixture

Blocking moiety: Same as in the 5 promoter oligonucleotide. The lengthening oligonucleotide:

TGTCGTGCAGCCTCCAGGACCCCCCCTCCCG GGAGAGCCATA (SEQ ID NO:

5); 12 pmol / reaction mixture

Blocking moiety: Same as promoter oligonucleotide

6. First capture probe:

GmGmGmĆmAmCmUmCmGmCmAmAmGmCmAmmCmCmCmU mlii 'AAAA AAAAAAAAAAA AAAAAAAAAAAAAAA (SEQ ID NO: 6); 3 pmole / reaction mixture

7. Second capture probe:

CmAjnUmGmGmUmGmCrnAmCmGroGinUrnCnrtJmAniCmGmTTT AAAAAAA AAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NO: 7); 3 pmole / reaction mixture

8. Detection probe:

CmGmUmUmCmCmGmCmAmGmAmCmCmAmCmUroAmUtn (Linker) GniAinAm CmGm (SEQ ID NO: 8): 4 pmole / reaction mixture

Probe Type: Molecular torch

Linker: 9-O-Dimethoxytrityltriethylene glycol, 1 - [(2-cyanoethyl) - (N, N-diisopropyl)] phosphoramidite (Glen Research Corporation, Sterling, VA; cat. No. 10-1909-90) 5'-Tag: 6-Carboxyfluorescein (FAM) (BioGenex, San Ramon, CA; cat. No. BGX-3008-01)

3'-Tag: 4- (4'-dimethylaminophenylazo) benzoic acid (DABCYL) (Prime Synthesis, Inc., Aston, PA)

II. REAGENTS AND OTHER INFORMATION ON THE PROTOCOL [0244]

1. Reagent Reagent. "Amplification Reagent" or "AMP Reagent" 20 contained 11.6 mM Trizma® based buffer in base form, 15 mM Trizma® hydrochloride based buffer, 25 mM MgCl2, 23.3 mM KCl2, 3.33% (vol ./vol.) glycerol,

0.05 mM zinc acetate, 0.76 mM dATP, 0.76 mM dCTP, 0.76 mM dGTP, 0.76 mM d'TTP, 0.02% (v / v) ProClin 300 preservative (Supelco, Bellefonte, PA; cat. No.

48126), 6.0 mM ATP, 6.0 mM CTP, 6.0 mM GTP, and 6.0 mM UTP, pH 7.81 to 8.0 at 22 ° C.

2. Enzyme Reagent. The "enzyme reagent" contained 70 mM N-acetyl L-cysteine, 10% (v / v) TRYTON® X-102 detergent, 16 mM HEPES, 3 mM EDTA, 0.05% (w / v) sodium azide , 20 mM Trizma® base buffer, 50 mM KCl2, 20% (v / v) glycerol, 165.6 mM trehalose, pH 7, and contained 224 RTU / ul of reverse transcriptase from Moloney 'mouse leukemia virus a ("MMLV") and 140 U / ul T7 RNA polymerase, with one unit (i.e. RTU or U) activity is defined as the synthesis or release of 5.75 fmol cDNA for 15 minutes at 37 ° C for MMLV reverse transcriptase and production of 5.0 fmol RNA transcript for 20 minutes at 37 ° C for T7 RNA polymerase.

3. Rinsing solution. "Wash Solution" contained 10 mM HERPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) ethyl alcohol, 0.02% (w / v) methyl paraben, 0.01 % (w / v) propyl paraben, 150 mM NaCl and 0.1% (w / v) sodium dodecyl sulfate, pH 7.5.

4. Transport center. The "transport medium" contained 150 mM HEPES, 8% (w / v) lithium lauryl sulfate and 100 mM ammonium sulfate, pH 7.5.

5. Target Capture Reagent. "Target Capture Reagent" or "TCR" contained the ingredients listed below. Additional information on the composition of this mixture is described below in the procedure for directed capture reagent (IIIA). The concentrations given are the final concentrations of the components when combined with the magnetic particle solution. The magnetic particles were Sera-Mag MG-CM Carboxylate Modified ™ (Seradyn, Inc., Indianapolis, IN; cat. No. 24152105-050250), 1 micron, super-paramagnetic particles covalently bound to 5 'amino modified oligo (dT). The components HEPES, lithium hydroxide, lithium chloride, EDTA, lithium lauryl sulfate and ammonium sulfate were introduced in the TCR solvent and transport medium.

First capture probe; 15.0 nM

Second capture probe; 15.0 nM

Tagged priming oligonucleotide; 60.0 nM

Terminating oligonucleotide; 4.0 nM

HEPES, free acid, dihydrate; 118.7 mM

Lithium hydroxide monohydrate; 98.9 mM

Lithium chloride, high purity; 470.6 mM

EDTA, free acid; 25.0 mM

Lithium lauryl sulfate; 110.2 mM Ammonium sulfate; 37.5 mM

Magnetic particles Seradyn Poly dT14; 0.075 μg / μl

6. Transcription Buffer. The "transcription buffer" contained 0.2% lithium lauryl sulfate.

7. Transcript used. HCV transcript

8. Product numbers for specific materials or equipment used.

KingFisher ™ plate (Thermo Labsystems, Franklin, MA; cat. No. 97002540)

MJ Research microtiter plate (Bio-Rad Laboratories, Inc., Hercules, CA; cat. No. HSP-9665)

Incubator Solo HT (Thermo Labsystems, Franklin, MA; cat. No. 5161580)

KingFisher ™ Comb instrument (Thermo Labsystems, Franklin, MA; catalog number 97002510) Eppendorf® Thermomixer R (Eppendorf North America; Westbury, NY; catalog number 022670107 or 022670158)

DNA Engine Opticon® 2 real-time PCR detection system (Bio-Rad Laboratories, Inc., Hercules, CA; cat. No. CFB-3220)

9. Additional information about the protocol.

For the experiments described in step B6 below, to each 2.0 ml microtube 3.3 μΐ target transcription buffer was added. The labeled priming oligonucleotide and terminating oligonucleotide were in water before being added to 2.0 mL microtubes. Samples were mixed using a vortex device for about 5 seconds. Incubation for 10 minutes at 60 ° C was generally sufficient for transcript uptake. The plates were kept at room temperature for 5 minutes after a 10-minute incubation to allow the plates to cool before the targeted uptake steps. At this point, the plates were transferred from the Solo HT incubator to a KingFisher system. The thermomixer speed was 1,400 rpm.

III. DIRECTED EXTRACTION PROTOCOL

A. Procedure for directed capture reagent (TCR).

[0245] The magnetic beads were slowly mixed at room temperature (RT) for 45 minutes and 150 µL of magnetic beads were added to 5 mL of TCR solvent (15 µg beads / reaction mixture when 50 µL was used per sample). The solution was slowly stirred at room temperature for 35 minutes, then a capture probe (to a final concentration of 0.12 pmol ^ 1 (6 pmol / 50 μl reaction mixture) was added to 5 ml TCR solvent.

B. Preparation of the sample.

[0246] An AMP reagent containing a promoter oligonucleotide, extension oligonucleotide and primer oligonucleotide (volume = 1600 Lii) was prepared. The solution was mixed using a vortex device and placed at 2-8 ° C until needed. The detection probe was prepared in an enzyme reagent and placed at 2-8 ° C until needed. Target dilutions were prepared in 0.2% LLS. 50 μl TCR was transferred to 200 μl wells on a microplate. Target at each copy level, labeled priming oligonucleotide and terminating oligonucleotide were added to 1.2 mL 50% transport medium, 50% H2O in 2.0 mL microtubes. Target samples were mixed using a vortex device and 150 μl transferred to a 200 μl well on a microplate (Plate 1) containing 50 μl TCR (each well did not contain or contained 1 million copies of HCV transcript and corresponding amounts of labeled priming and terminating oligonucleotide).

C. Target Capture Protocol [0247] 200 μl microplate (Plate 1) were incubated at 60 ° C for 10 minutes using a Labsystems Solo HT incubator (Plate 1), and then the microplate was placed at RT for 5 minutes (Plate 1). 200 μl microplate (Plates 2 and 3) were prepared using 200 μl washing reagent. An amplification plate (4-MJ research 96-well microtiter plate) was prepared using 30 μl AMP reagent per well. The 96-well comb was placed in Plate 1. All four plates were loaded onto KingFisher 96 unit and the targeted capture protocol was started as follows.

[0248] The contents of Plate 1 were mixed for 5 minutes at very low speed and the balls were collected for 12 counts, and then released into Plate 2 for 10 seconds at low speed. Then the contents of Plate 1 were mixed for 1 second using very low speed, the balls were collected for 12 counts and the balls were released into Plate 2 for 10 seconds using low speed.

[0249] The contents of Plate 2 were mixed for 30 seconds at medium speed and the balls were collected for 12 counts and then released onto Plate 3 for 10 seconds using very low speed. The contents of Plate 2 were then mixed for 1 second at very low speed and the beads were collected for 12 counts and released onto Plate 3 using very low speed.

[0250] The contents of Plate 3 were mixed for 30 seconds at medium speed, the beads were collected for 12 counts and the balls were released into Plate 4 for 10 seconds using medium speed. Plate 3 contents were then mixed for 1 second at very low speed, balls were collected for 12 counts and released onto plate 4 for 10 seconds using medium speed.

[0251] The 96-well microtiter plate (Plate 4) was removed and transferred to the table, covered with a sealing layer and placed in a DNA Engine Opticon® 2 real-time PCR detection system (Bio-Rad Laboratories; Hercules, CA) ("device for real-time testing ").

D. TMA in real time.

[0252] Real-time TMA was performed as follows. The plate was incubated for 5 minutes at 42 ° C, then removed and placed at 42 ° C in a thermomixer. A 10 Pl aliquot of enzyme reagent was added to each reaction well. The microtiter plate was covered with a sealing adhesive tape, gently shaken for 30 seconds in a thermomixer and then placed on a 42 ° C real-time testing device, where real-time monitoring of the test was started. T time values ​​that served as indicators of the amount of synthesized amplicon were determined based on the monitored fluorescence signals. See Light et al., US Patent Application Publication No. US 2006-0276972, paragraphs 506549.

IV. RESULTS AND CONCLUSIONS [0253] Experiments were performed according to the procedures described above for detecting HCV transcript (8 replicates). The TCR contained the same priming oligonucleotide in each study. A directed capture step was performed to bind the HCV transcript and remove unhybridized tagged priming oligonucleotide and terminating oligonucleotide. After the targeted uptake step, the AMP reagent was contacted with TCR beads, the AMP reagent containing a primer oligonucleotide specific for complement of the tag sequence. This step did not include any tagged priming oligonucleotide.

[0254] 8 replicates were performed for each set of conditions. The detection probe was added in the enzyme reagent at a concentration of 4 pmole per reaction mixture. The HCV AMP reagent contained 12 pmol of promoter oligonucleotide, 12 pmol of extension oligonucleotide and 12 pmol of priming oligonucleotide per reaction mixture.

[0255] In the first set of experiments, reaction results were compared in which no copies of HCV transcript to TCR or AMP reagent were given with reactions in which 1 x 10 was given.⁶ copies of the HCV transcript to TCR. Figure 17 shows the raw curves for HCV amplifications in which no target was given to the AMP reagent. There was no detectable amplification when the HCV transcript was not administered to the TCR or AMP reagent, while the average time T for reactions containing 1 x 10⁶ copy of the HCV transcript in the TCR was 6.3 minutes. The "time T" values ​​refer to the time the signal appeared (the time the signal rises above the background), and a summary of these values ​​for the experiments performed is provided in Table 1 below.

[0256] In a second set of experiments, reaction results were compared in which 1 x was given

10⁶ copies of HCV transcript only to AMP reagent with reactions in which 1 x 10 is given⁶ copies of the HCV transcript to TCR only. Figure 18 shows the raw curves for HCV amplifications in which the target was administered to the AMP reagent. There was no detectable amplification when the HCV transcript was administered to the AMP reagent, while the average time T for reactions containing 1 x 10⁶ copy of the HCV transcript in the TCR was

6.3 minutes (Table 1). In the absence of a target in TC samples, it did not amplify even in the presence of one million copies of HCV transcript administered to the AMP reagent. [0257] A third set of experiments compared the results of reactions in which 1 x 10 was supplied to the AMP reagent⁶ copy of the HCV transcript and labeled priming oligonucleotide (no copy of the HCV transcript in the TCR) with the results of the reaction in which 1x10 was delivered to the TCR⁶ copy of the HCV transcript, and a tagged priming oligonucleotide was provided to the AMP reagent. Figure 19 shows that the mean the T time for the million copies of the HCV transcript present only in the targeted uptake step with the labeled priming and terminating oligonucleotide administered to the AMP reagent was 7.2 minutes. For samples with target, terminating oligonucleotide and labeled priming oligonucleotide administered to the AMP reagent, strong amplification was also obtained from sometimes T = 8.6 minutes (Table 1).

Table 1. Summary of T-times (Avg. T-times and standard deviations (SD) of T-times)

<td>Sample ID</td><td>Name to</td><td>The amount of purpose</td><td>Sum</td><td>RN1</td><td>TN1</td><td>Avg. Time T</td><td>SD Time T.</td>

<td>1 million target in TC-x6.0</td><td>HCV</td><td>1E6</td><td> 8</td><td> 7</td><td> 8</td><td> 6,3</td><td> 0,11</td>

<td>1 million target in TC, tagged non-T7 primer and amp-x6.0 terminating oligonucleotide</td><td>HCV</td><td>1E6</td><td> 8</td><td> 8</td><td> 8</td><td> 7,2</td><td> 0,20</td>

<td>1 million target in TC-x6.0</td><td>HCV</td><td>1E6</td><td> 8</td><td> 8</td><td> 7</td><td> 6,3</td><td> 0,05</td>

<td>no target in TC, 1 million target in amp-x0.0</td><td>HCV</td><td> 0,00</td><td> 8</td><td> 8</td><td> 0</td><td>ON</td><td>ON</td>

<td>no target in TC, 1 million target, labeled non-T7 primer and amp-x0.0 terminating oligonucleotide</td><td>HCV</td><td> 0,00</td><td> 8</td><td> 8</td><td> 8</td><td> 8,6</td><td> 0,21</td>

<td>no target in Tc-x0.0</td><td>HCV</td><td> 0,00</td><td> 8</td><td> 8</td><td> 0</td><td>ON</td><td>ON</td>

[0258] The results of these experiments show that only when the tagged priming oligonucleotide was present in the AMP reagent together with the priming oligonucleotide, zero TCR samples were amplified when 1 million copies of the HCV transcript were administered to the AMP reagent. Thus, HCV transcript entering the system in the AMP reagent is not amplified unless labeled priming oligonucleotides are also provided in the AMP reagent.

[0259] The above Example demonstrates how a tagged priming oligonucleotide that hybridized to an HCV template can be used to selectively detect HCV nucleic acids in an interesting sample without interference from contaminating nucleic acids introduced after the targeted capture step. The next example illustrates how a similar approach was used to detect bacterial nucleic acids in the sample of interest, despite the presence of contaminating matrices in the reagents used to carry out the amplification reaction. Preferably, the uncomplexed tagged priming oligonucleotide was substantially absent in the reaction mixture when the complex comprising the tagged priming oligonucleotide and the template was contacted with the DNA polymerase used in the amplification reaction.

[0260] Example 2 below describes two procedures for the amplification of E. coli rRNA nucleic acids, which procedures differed in using both labeled priming oligonucleotide and targeted uptake. In the first procedure, an unlabeled E. coli specific primer oligonucleotide was used in combination with the terminating oligonucleotide, promoter oligonucleotide, and detection probe. The second procedure procedure uses a tagged priming oligonucleotide containing a target complementary sequence identical to that contained in the unlabeled E. coli specific primer oligonucleotide of the first procedure, a tag specific priming oligonucleotide, as well as a terminating oligonucleotide, a promoter oligonucleotide and detection. A tag specific priming oligonucleotide that had a nucleotide sequence corresponding to the HIV-1 segment hybridized to the complement of the tag sequence contained in the tagged priming oligonucleotide, but did not hybridize to the E. coli template nucleic acid rRNA or its complement. For the second procedure, the terminating oligonucleotide, promoter oligonucleotide and detection probe were identical to those used in the first procedure. As shown below, amplification reactions that bypassed the tagged priming oligonucleotide did not distinguish between samples containing 0 and 10⁶ copies of synthetic target rRNA from E. coli. In contrast, in an approach involving the use of a labeled primer and targeted oligonucleotide₃ the uptake clearly distinguishes samples containing 0 and 10³ copies of synthetic target rRNA from E. coli.

Example 2

USE OF LABELED STARTER OLIGONUCLEOTIDE ALLOWS DISTINCTION BETWEEN SAMPLE OR EXTREME MATRIXES

A. AMPLIFICATION USING AN UNLABELED STARTER OLIGONUCLEOTIDE WITHOUT DIRECTED UCHESTRATION [0261] In the first procedure, amplification reactions were carried out using a synthetic E. coli rRNA template using an unlabeled primer oligonucleotide that hybridized the oligonucleotide of the nucleotide and the nucleotide of the oligonucleotide of the oligonucleotide, which hybridized to the oligonucleotide of the oligonucleotide, which hybridized to the oligonucleotide of the nucleotide, and the nucleotide of the oligonucleotide hybridized oligonucleotide, which has a hybridized oligonucleotide and oligonucleotide nucleotide hybridized oligonucleotide, which has a hybridized oligonucleotide nucleotide, which has a nucleotide oligonucleotide ligand hybridized oligonucleotide nucleotide, torch. Reactions were started by adding the synthetic matrix directly to the reaction mixtures (i.e. without prior purification by targeted uptake) in an amount of 0 or 10⁶ copy / reaction mixture. A molecular torch detection probe was used to monitor amplicon production as a function of time. In the nucleotide sequences set out below, modifications to the polynucleotide backbone by 2'-O-methyl ribose (2'-O-Me) are indicated by a small "m". Blocking moieties on the 3'-ends of the promoter oligonucleotide and the terminating oligonucleotide contained a 3'-to-3 'linker, which was prepared using 3'-dimethyltrityl-N-benzoyl-2'-deoxycytidine, 5'-succinyloyl (long chain alkyl) -amino-CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). Oligonucleotides, reagents and the most important methods used in the procedure were as follows.

I. OLIGONUCLEOTIDES:

[0262]

1. Unlabeled primer oligonucleotide:

CmUmGmCmTGGCACGGAGTTAGCCGGTGCTTC (SEQ ID NO: 9)

2. Promoter oligonucleotide:

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG blocking moiety (SEQ ID NO: 10)

3. The terminating oligonucleotide:

GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-blocking moiety (SEQ ID NO: 11)

4. Detection probe:

'CmUmGmCmGmGmGmUniAmAmCinGniUinCinAmAinUmGmAinGjnCmAni AmAm²CGCAG³ (SEQ ID NO: 12) ¹ fluorescein ² C9 connector ³ DABCYL

5. Synthetic E. coli rRNA template:

AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCC

TAACACATGCAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGA

GTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAAC

TACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGA

CCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGG

GGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGC

CACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGA

ATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAA

GGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAAT

ACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCA

GCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTA

AAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCT

GGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATT

CCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAG

GCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAA

CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTT

GTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGG

AGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGC

GGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTG

ACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGTGAGACA

GGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG

CAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAA

GGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCA

TGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGA

AGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATT

GGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAG

AATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT

GGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTAC

CACTTTGTGATTCATGACTGGGGTGAAGTCGTAACAAGGTAACCGTAGGGGAA

CCTGCGGTTGGATCACCTCCTTA (SEQ ID NO: 13)

II. REAGENTS AND OTHER PROTOCOL INFORMATION:

[0263]

Amplification reagents and enzymes were essentially as described in the Example

1. The procedures using the unlabeled primer oligonucleotide that hybridized to the E. coli template did not include oligonucleotides or targeted uptake reagents, did not include a transport medium or a scrubbing solution, nor did it include an extension oligonucleotide.

A. Real-time amplification protocol.

[0264] Sample solutions were prepared using a primer-free amplification reagent, unlabeled priming oligonucleotide, promoter oligonucleotide, terminating oligonucleotide, detection probe, and synthetic template nucleic acid. A 30 μΐ aliquot of prepared sample solution was added to each well from the 96-well microtiter plate. The microtiter plate was covered with sealing adhesive tape, incubated initially for 10 minutes at 60 ° C in a real-time DNA ENGINE OPTICON® 2 temperature control device (Bio-Rad Laboratories; Hercules, CA) and then at set temperature at 42 ° C for 5 minutes. The plate was then removed from the real-time measurement device and placed at 42 ° C in the thermomixer. A 10 Pl aliquot of enzyme reagent was added to each reaction well. The microtiter plate was covered with a sealing adhesive tape, gently shaken for 30 seconds in a thermomixer, and then placed in a real-time measurement device at 42 ° C, where real-time test monitoring began. T time values ​​that served as indicators of the amount of synthesized amplicons were determined based on the monitored fluorescence signals.

III. RESULTS AND CONCLUSIONS [0265] As indicated in Figure 20, substantially the same results were observed in reactions that included 0 or 10⁶ template nucleic acid copy, so the test showed no distinction between these two conditions. More specifically, fluorescence signals indicating the formation of nucleic acid amplification products

E. coli went beyond background signals at substantially similar times (i.e., T = 31.74 minutes at 0 copy and 31.19 minutes at 10⁶ copy) in both reactions. Thus, the real-time amplification profile characteristic of high levels of the nucleic acid template was obtained even in the absence of the added E. coli rRNA template. This was consistent with the presence of contaminating bacterial nucleic acid matrices in one or more reagents used to carry out the amplification reaction according to the targeted capture procedure.

B. AMPLIFICATION USING LABELED STARTER OLIGONUCLEOTIDE AND DIRECTIONAL ADMISSION [0266] In the second procedure, a labeled priming oligonucleotide and a targeted uptake step were used to perform the amplification reaction using 0, 10 samples tested³ or 10⁵ copy of synthetic transcript from E. coli. Oligonucleotides used in this procedure are indicated below. A molecular torch detection probe was added as a component of the enzyme reagent. After targeted uptake, the labeled priming oligonucleotide that did not hybridize to the template nucleic acid was removed from the system in standard directed uptake and wash steps. The complex including the rRNA template and the tagged priming oligonucleotide remained captured on the super-paramagnetic particles. Amplification reactions were carried out using essentially such reagents as described above, in addition to being replaced by the capture probe with the non-specific targets of the sequence-specific capture probes used in Example 1. Amplification reactions were performed in duplicate and monitored using a molecular torch detection probe, essentially as described in Example 1, except that the extension oligonucleotide was omitted. As above, the modification of the main chain of the polynucleotide by 2'10 O-methyl ribose (2'-O-Me) in the sequences shown below are indicated by the small "m". Blocking moieties at the 3'-ends of the promoter oligonucleotide and the terminating oligonucleotide contained a 3'-to-3 'linker, which was prepared using 3'dimethyltrityl-N-benzoyl-2'-deoxycytidine, 5'-succinyloyl (long chain alkyl) amino-CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). The oligonucleotides, reagents and the most important methods used in the procedure were as follows.

I. Oligonucleotides:

[0267]

1. Tagged priming oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCG GTGCTTC (SEQ ID NO: 14)

2. Tag specific primer oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter oligonucleotide:

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG blocking moiety (SEQ ID NO: 10)

4. The terminating oligonucleotide:

CmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-blocking moiety (SEQ ID NO: 11)

5. Non-specific capture probe:

KjnKmKmKmKjwtfCnaCjnKm ^^

AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16)

6. Detection probe:

'CmUmGmCmGmGraGmUniAniAmCmGmUmCmAmAmUmGmAmGinCmAm AmAnfCGCAG³ (SEQ ID NO: 12) ¹ fluorescein ² C9 connector ³ DABCYL

7. Synthetic E. coli rRNA template (See above)

II. REAGENTS AND OTHER PROTOCOL INFORMATION [0268] Reagents and experimental protocols were essentially as described in Example 1, with the replacement by a non-specific oligonucleotide for targeted uptake of the first and second oligonucleotide for directed uptake, replacing by the above-described E. coli oligonucleotide specific oligonucleotides specific for HCV and omitting the lengthening oligonucleotide.

III. NON-SPECIFIC DIRECTED RUNNING PROTOCOL

A. Preparation of the directed capture reagent (TCR).

[0269] The starting mixture of magnetic beads was stirred at room temperature for 30 minutes. An aliquot of approximately 150 μl of magnetic bead suspension was added to 5 ml of TCR diluent (15 μg beads / reaction mixture using 50 μl / sample), followed by slow stirring at room temperature for 30 minutes. Then, non-specific oligonucleotide for targeted uptake was added to 5 ml of the TCR mixture to give a final concentration of 0.12 pmol / μΙ. The prepared TCR was gently mixed at room temperature until needed.

B. Preparation of the sample.

[0270] The amplification solution was prepared using the primer amplification reagent, promoter oligonucleotide and tag specific priming oligonucleotide. The prepared amplification solution was mixed using a vortex device and then kept at 2-8 ° C until needed. The enzyme reagent containing the molecular torch detection probe was then prepared and kept at 2-8 ° C until needed. Matrix rRNA dilution was prepared in 0.2% LLS (lithium lauryl sulfate). Portions (50 µL of magnetic bead solution for directed capture were transferred to wells on a microtiter plate for a KINGFISHER 96 magnetic particle processing device (Thermo Fisher Scientific, Inc.; Waltham, MA). Samples of diluted matrix, labeled priming oligonucleotide and terminating oligonucleotide were added then to 1.5 ml 50% transport medium diluted with water. The target sample mixture was mixed using a vortex device, and 150 μl aliquots transferred to the wells of the microtiter plate (Plate 1) containing 50 μl of solution for directed capture (each well contained 0, 10³ or 10⁵ copy of E. coli transcript and the corresponding amount of labeled priming oligonucleotide and terminating oligonucleotide).

C. Targeted capture protocol.

[0271] First, a microtiter plate containing 200 μl of washing reagent (Plate 2) was prepared. Another microtiter plate (Plate 3) was prepared to perform the amplification reaction in which each well to be used for the reaction contained 30 μl of amplification reagent. All three plates (Plates 1-3) were loaded onto the magnetic ball processing unit. Magnetic beads carrying nucleic acid complexes were isolated from Plate 1, washed in Plate 2, and then transferred to Plate 3 using standard procedures known to those of ordinary skill in the art. Plate 3 was removed from the magnetic ball processing unit, covered with sealing adhesive tape, and then placed on a real-time temperature monitoring device.

D. Real-time amplification protocol.

[0272] Plate 3 was incubated at 42 ° C for 5 minutes in a real-time measurement device. The microtiter plate was then removed from the real-time measurement device and placed in a thermomixer at 42 ° C. A 10 Pl aliquot of the enzyme reagent containing the detection probe was added to each reaction well, and then covered with a sealing adhesive tape. The plate was gently shaken for 60 seconds in a thermomixer and then placed back into the 42 ° C real-time measurement device, where real-time test monitoring began. T time values ​​that served as indicators of the amount of synthesized amplicons were determined based on monitored fluorescence signals.

IV. RESULTS AND CONCLUSIONS [0273] Figure 21 graphically illustrates the benefits of the disclosed approach to nucleic acid amplification. In procedures using tagged primer oligonucleotides complementary to the target of interest, a target capture step and tag specific primer oligonucleotide that was not complementary to the target of interest (i.e. E. coli rRNA), significantly reduced levels of amplification were obtained₃ background, which made it easy to distinguish between 0 and 10 copies of bacterial template nucleic acid. More specifically, the mean T-time values ​​determined for reactions carried out using 10⁵ copies, 10³ copies and 0 copies of the E. coli matrix were 24.7 minutes, 30.6 minutes and 37.5 minutes, respectively. Together with the results shown in Figure 4, these findings were consistent with the presence of bacterial nucleic acids in common reagents used to perform in vitro nucleic acid amplification reactions. Still, the procedure using the tagged priming oligonucleotide was useful for the detection of E. nucleic acids. colas present in the test sample without interference from exogenous template nucleic acids provided in the amplification reagents. For example, a qualitative test for₃ detection of nucleic acids from E. coli at a level of 10 or more copies in the sample tested could depend on obtaining a threshold for the fluorescence signal, or the value of time T after a predetermined reaction time (e.g. 35 minutes).

[0274] The next example shows comparative results showing how two different detection probes affected the curve profiles for real-time amplification. The results also showed how a labeled approach can be used₃ a priming oligonucleotide to distinguish between 0 and 10³ a copy of the synthetic template nucleic acid from E. coli, which is approximately equal to the number of copies of 16S rRNA present in a single bacterium.

[0275] Example 3 describes the detection of E. coli rRNA templates in real-time amplification reactions using three different detection probes.

Example 3

ALTERNATIVE TORCH PROBE PROJECTS MAY IMPROVE THE TEST RESULTS [0276] Amplification reactions were performed and monitored in real time using one of three different detection probes. The synthetic matrix nucleic acid, non-specific capture oligonucleotide, labeled priming oligonucleotide, terminating oligonucleotide, promoter oligonucleotide and tag specific priming oligonucleotide used to carry out the reaction were identical to those used in the second procedure of the previous Example. The fragment hybridizing with the E. coli target of the tagged priming oligonucleotide corresponded to nucleotide positions 24-57 of SEQ ID NO: 14 (i.e. target hybridizing sequence corresponding to SEQ ID NO: 19). The fragment hybridizing with the E. coli target of the promoter oligonucleotide corresponded to nucleotide positions 27-47 of SEQ ID NO: 10 (i.e., target hybridizing sequence corresponding to SEQ ID NO: 20). Four replicates were performed for each condition. As before, the detection probe was added together with the enzyme reagent. Reagents and protocols for non-specific targeted uptake, sample preparation and real-time amplification were also essentially as described in the second procedure of the previous Example. In particular, the reaction was carried out using 0, 10³ or 10⁵ copy of synthetic E. coli matrix. As above, 2'-O-methyl-ribose (OMe) main chain modifications of the polynucleotide in the sequences below are indicated by a small "m". Blocking moieties at the 3'-ends of the promoter oligonucleotide and the terminating oligonucleotide contained a 3'-to-3 'linker made using 3'-dimethyltrityl-N-benzoyl-2'deoxycytidine, 5'-succinyl- (long chain alkyl) -amino -CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). Oligonucleotides, reagents and the most important methods used in the procedure were as follows.

I. OLIGONUCLEOTIDES:

[0277]

1. Tagged priming oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCG GTGCTTC (SEQ ID NO: 14)

2. Tag specific primer oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter oligonucleotide :

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG blocking moiety (SEQ ID NO: 10)

4. The terminating oligonucleotide:

GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm-blocking moiety (SEQ ID NO: 11)

5. Non-specific capture probe:

KmKniKniKmKnikniKroKTOKmKiriKmKmKinKinKmKinKmKinTTTAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16)

6. Detection probe:

2 3 ¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCm²GmCmUmCmGm³(SEQ ID NO: 17) 'CmGmAnjGmCraAmAmAinGniGmUmAmUmUniAniAniCinUmUraUniAmCmU mCm²GrnCmUrnCmGn? (SEQ ID NO: 18)¹ fluorescein ² C9 connector ³ DABCYL

7. Synthetic E. coli rRNA template (See above)

II. REAGENTS AND OTHER PROTOCOL INFORMATION [0278] Reagents and experimental protocols were essentially as described in Example 2, with a slight change in the conditions used for targeted uptake.

III. NON-SPECIFIC DIRECTED RUNNING PROTOCOL:

A. Preparation of the directed capture reagent (TCR).

[0279] The initial suspension of the magnetic beads was mixed at room temperature for 25 minutes. A 150 μΐ aliquot of the magnetic bead suspension was added to 5 ml TCR diluent (15 μg beads / reaction mixture using 50 μl / sample), followed by slow stirring at room temperature for 25 minutes. Then, the non-specific capture oligonucleotide was added to 5 ml of the TCR mixture to give a final concentration of 0.12 pmol / μΐ. The prepared TCR was gently mixed at room temperature until needed.

B. Preparation of samples.

[0280] Amplification solutions were prepared using a primer-free AMP Reagent, a promoter oligonucleotide, and a tag specific priming oligonucleotide. Prepared amplification solutions were mixed using a vortex device and then kept at 2-8 ° C until needed. Then enzyme reagents containing molecular torch detection probes were prepared and kept at 2-8 ° C until needed. Matrix rRNA dilutions were prepared in 0.2% LLS as described above. Portions (50 μΡ of magnetic bead solution for directed capture were transferred to wells on a microtiter plate for a KINGFISHER 96 magnetic bead processing device (Thermo Fisher Scientific, Inc.; Waltham, MA). Samples of diluted matrix, labeled priming oligonucleotide and terminating oligonucleotide were then added to 1.5 ml 50% transport medium diluted with water. The target containing sample mixture was mixed using a vortex device and 150 μl aliquots transferred to wells on a microtiter plate (Plate 1) containing 50 μl of solution for directed capture (each well contained 0, 10 or 10 copies of E. coli transcript and the corresponding amount of labeled priming oligonucleotide and terminating oligonucleotide).

C. Targeted capture protocol.

[0281] The microtiter plate (Plate 1) was incubated at 60 ° C for 15 minutes using a SOLO HT incubator (Thermo Labsystems; Franklin, MA). The microtiter plate was then placed on the table at room temperature and allowed to equilibrate for 5 minutes (Plate 1). Then a second microtiter plate containing 200 μl of washing reagent (Plate 2) was prepared. A third microtiter plate (Plate 3) was prepared for performing the amplification reaction in which each well to be used in the reaction contained 30 μl of reagent for

100 amplification. All three plates were loaded onto the magnetic particle processing unit. Magnetic beads carrying nucleic acid complexes were isolated from Plate 1, washed in Plate 2, and then transferred to Plate 3 using standard procedures known to those of ordinary skill in the art. Plate 3 was removed from the magnetic particle treatment unit, covered with sealing adhesive tape, and then placed in a real-time temperature monitoring device.

D. Real-time amplification protocol.

[0282] Plate 3 was incubated in a real-time measurement device at 42 ° C for 5 minutes. The microtiter plate was removed from the real-time measurement device and placed in a thermomixer at 42 ° C. A 10 Pl aliquot of the enzyme reagent containing the detection probe was added to each reaction well, and then covered with a sealing adhesive tape. The plate was gently shaken for 60 seconds in a thermomixer and then placed back into the 42 ° C real-time measurement device where real-time test monitoring began. T time values, which served as indicators of the amount of synthesized amplicons, were determined on the basis of monitored fluorescence signals.

IV. RESULTS AND CONCLUSIONS [0283] The results presented in Table 2 summarize the average time values ​​T (column

3), and standard deviations from the average time T values ​​(column 4) for reactions carried out with different detection probes. The summary in the table confirmed that very good results in real-time tests were obtained using all of the tested detection probes. Each probe preferably gave a very low signal at 0 copy of the input target. More specifically, the amplicon detected in the reactions carried out using the 0 input copy of the synthetic matrix was essentially undetectable when the reactions involved detection probes with SEQ ID NO: 17 and SEQ ID NO: 18. Thus, reactions that included one of the detection probes identified as SEQ ID NO: 17 and SEQ ID NO: 18 gave excellent results that enabled easy detection of template nucleic acids corresponding to approximately the amount contained in a single bacterium.

Table 2. Use of alternative detection probes to improve discrimination in the test

Quantity of sensor Detection probe (copies) avg. time T (minutes)

SD time T (minutes)

101

<td>The amount of matrix</td><td>Detection probe</td><td>Wed. time T</td><td>SD time T.</td>

<td>(copies)</td><td></td><td>(Minutes)</td><td>(Minutes)</td>

<td> 0</td><td></td><td>ON</td><td>ON</td>

<td>₁₀³</td><td>SEQ ID NO: 17</td><td> 38,2</td><td> 2,81</td>

<td>₁₀⁵</td><td></td><td> 26,4</td><td> 0,32</td>

<td> 0</td><td></td><td>ON</td><td>ON</td>

<td>₁₀³</td><td>SEQ ID NO: 18</td><td> 35,9</td><td> 2,33</td>

<td>₁₀⁵</td><td></td><td> 28,8</td><td> 0,45</td>

[0284] Considering the results set out in Examples 2 and 3, each of SEQ ID NO: 12 and 17-18 is a preferred molecular torch probe for detecting E. coli using the methods described herein. Highly preferred probes useful for detecting E. coli nucleic acids will have target complementary sequences corresponding to nucleotide positions 2-24 contained in SEQ ID NO: 12 (i.e. target hybridizing sequence corresponding to SEQ ID NO: 21) or nucleotide positions 2-17 contained in SEQ ID NO: 17 (i.e. target hybridizing sequence corresponding to SEQ ID NO: 22), or nucleotide positions 2-24 contained in SEQ ID NO: 18 (i.e., target hybridizing sequence corresponding to SEQ ID NO: 23). In general, probes useful for detecting E nucleic acids. coli will have target hybridizing sequences comprising at least 16 consecutive nucleotides contained in the sequence TGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTC (SEQ ID NO: 24). The total preferred lengths of the desired probes will be up to 39 nucleotides, more preferably up to 29 nucleotides, more preferably up to 23 nucleotides, or even more preferably up to 16 nucleotides. Of course, useful probes may contain equivalent RNA and DNA bases and include additions to the above described probes.

NEXT ASPECTS [0285] Further aspects of the present disclosure are set out in the following numbered paragraphs:

1. A method of selectively amplifying at least one target nucleic acid sequence from a nucleic acid sample, the method comprising the steps of:

(a) treating the target nucleic acid sequence in the nucleic acid sample with a heterologous tag sequence to form a tagged target nucleic acid sequence;

(b) reducing in this sample the effective concentration of heterologous tag sequences that have not become part of this tagged target nucleic acid sequence and are in a form capable of producing a tagged target nucleic acid sequence with that target nucleic acid sequence; and (c) exposing said labeled target nucleic acid sequence to reagents and conditions sufficient for detectably amplifying said target nucleic acid sequence, wherein after step (b) at this stage of exposure, said nucleic acid sample is exposed to a known contaminating source of that target acid sequence nucleic and wherein the detectable amplification of this target nucleic acid sequence is essentially limited to the amplification of the target nucleic acid sequence provided in said tagged target nucleic acid sequence from step (a), and not the target nucleic acid sequence provided from this known contaminant source.

2. The method of paragraph 1, wherein one or more of these reagents are made of a material that is known to be a contaminating source for this target nucleic acid sequence.

3. The method of paragraph 2, wherein one or more of these reagents is prepared using a microorganism containing this target nucleic acid sequence.

4. The method of paragraph 3, wherein one or more of these reagents contains at least one nucleic acid polymerase.

5. The method of paragraph 1, wherein one or more components used in the method other than one or more of these reagents comprises a known contaminating source for that target nucleic acid sequence.

6. The method of paragraph 1, wherein the environmental conditions for this method include a known contaminating source for this target nucleic acid sequence.

7. The method of paragraph 1, wherein during step (b) this tagged target nucleic acid sequence is immobilized on a solid support.

8. The method of paragraph 1, wherein step (b) comprises removing heterologous tag sequences that have not become part of this tagged target nucleic acid sequence from this nucleic acid sample.

9. The method of paragraph 1, wherein step (b) comprises inactivating heterologous tag sequences that have not become part of this tagged target nucleic acid sequence to form an inactivated heterologous tag sequence.

10. The method of paragraph 9, further comprising removing said inactivated heterologous tag sequence from this nucleic acid sample during step (b).

103

11. The method of paragraph 1, wherein said heterologous tag sequence is contained in a tagged oligonucleotide, which tagged oligonucleotide comprises a first and a second region, said first region comprising a target hybridizing sequence that hybridizes to the 3'-end of said target acid sequence nucleic acid, and the latter region comprises a tag sequence located 5 'to that of the target hybridizing sequence, and wherein said tag sequence does not stably hybridize to a target nucleic acid comprising that target nucleic acid sequence.

12. The method of paragraph 1, wherein said target hybridizing sequence is a universal oligonucleotide.

13. The method of paragraph 8, wherein during step (a) this heterologous tag sequence is in an active form that allows that heterologous tag sequence to produce that tagged nucleic acid sequence, and wherein in step (b) that heterologous tag sequence that did not form this the labeled target nucleic acid sequence is converted into an inactive form, which prevents this heterologous tag sequence from producing a tagged target nucleic acid sequence during step (c).

14. The method of paragraph 1, wherein step (c) comprises producing amplification products in a nucleic acid amplification reaction using the first and second oligonucleotides, said first oligonucleotide comprising a sequence that hybridizes to the 3'-end of this complement of this target nucleic acid sequence and the second oligonucleotide comprises a sequence that hybridizes to the complement of this tag sequence, but does not stably hybridize to this target nucleic acid sequence, each of these amplification products having a base sequence that is substantially identical or complementary to the base sequence of that target nucleic acid sequence, and further comprising a base sequence that is substantially identical or complementary to all or part of this tag sequence.

15. The method of paragraph 14, wherein these conditions are isothermal.

16. The method of paragraph 15, wherein said target nucleic acid sequence is amplified by a transcription based amplification reaction.

17. The method of paragraph 16, wherein said transcription-based amplification reaction is a TMA reaction.

18. The method of paragraph 14, wherein said amplification reaction is a PCR reaction.

19. The method of paragraph 1, wherein said target nucleic acid sequence is contained in the nucleic acid of a single species of microorganisms.

104

twenty. The method of paragraph 1, wherein said target nucleic acid sequence is contained in the nucleic acid of many species of microorganisms.

21. The method of paragraph 1, which method is selective for the amplification of the target nucleic acid sequence contained in each of the numerous target nucleic acids, and wherein in step (a) this heterologous tag sequence produces a tagged target nucleic acid sequence with that target nucleic acid sequence from each of the numerous target nucleic acids present in this nucleic acid sample.

22. The method of paragraph 21, wherein said target nucleic acid sequence contained in each of these multiple target nucleic acids is the same nucleic acid sequence.

23. The method of paragraph 1, wherein at least a portion of this nucleic acid sample is obtained from a clinical, water, industrial, environmental, seed, beverage or food source.

24. The method of paragraph 1, which method is a sterility test.

25. The method of paragraph 24, which method is a method of diagnosing sepsis.

26. A method of selectively amplifying at least one target nucleic acid sequence from a nucleic acid sample, the method comprising the steps of:

(a) treating a nucleic acid sample comprising the target nucleic acid sequence with a tagged oligonucleotide comprising the first and second regions, said first region comprising a target hybridizing sequence that hybridizes to the 3'-end of said target nucleic acid sequence and the second region having takes the tag sequence located 5 'to that of the target hybridizing sequence, wherein the second region does not stably hybridize to a target nucleic acid comprising this target nucleic acid sequence;

(b) reducing in this nucleic acid sample the effective concentration of unhybridized tagged oligonucleotide being in an active form in which the target hybridizing sequence from that unhybridized tagged oligonucleotide is available for hybridization with that target nucleic acid sequence; and (c) producing amplification products in a nucleic acid amplification reaction using the first and second oligonucleotides, said first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-end of the complement of this target nucleic acid sequence, and said second oligonucleotide comprising the sequence hybridizing, which hybridizes to the complement of this tag sequence, wherein the second oligonucleotide does not stably hybridize with this target nucleic acid, and wherein each of these amplification products comprises a base sequence that is substantially identical or complementary to the base sequence of that target nucleic acid sequence and furthermore comprises a base sequence that is substantially identical or complementary to all or a portion of this tag sequence.

27. The method of paragraph 26, wherein step (b) comprises removing the unhybridized tagged oligonucleotide from this nucleic acid sample.

28. The method of paragraph 27, wherein during step (b) this target nucleic acid sequence is immobilized on a solid support.

29. The method of paragraph 26, wherein step (b) comprises inactivating the unhybridized tagged oligonucleotide so that the unhybridized tagged oligonucleotide does not stably hybridize with this target nucleic acid sequence during step (c).

thirty. The method of paragraph 29, further comprising removing unhybridized tagged oligonucleotide from said nucleic acid sample during step (b).

31. The method of paragraph 29, wherein step (b) does not involve the use of an enzyme with nuclease activity.

32. The method of paragraph 29, wherein during step (a) said tagged oligonucleotide is in an active form that allows this target hybridizing sequence to hybridize with that target nucleic acid sequence, and wherein in step (b) the unhybridized tagged oligonucleotide is transformed in an inactive form that blocks or prevents hybridization of this tagged oligonucleotide with this target nucleic acid sequence during step (c).

33. The method of paragraph 32, wherein the conditions in steps (b) and (c) are less stringent than the conditions in step (a).

34. The method of paragraph 33, wherein the temperature of said nucleic acid sample is lowered between steps (a) and (b).

35. The method of paragraph 32, wherein in step (b) the unhybridized tagged oligonucleotide of step (a) is converted from a single-stranded form into a duplex form.

36. The method of paragraph 35, wherein the duplex form is a hairpin tag molecule comprising a tag closing sequence connected to the 5'-end of said tagged oligonucleotide, which tag closing sequence hybridizes with that target hybridizing sequence under conditions step (b), block 106 thereby hybridizing the unhybridized tagged oligonucleotide of step (a) with that target nucleic acid sequence in steps (b) and (c).

37. The method of paragraph 36, wherein said tag closing sequence is joined to said tagged oligonucleotide via a non-nucleotide linker.

38. The method of paragraph 37, wherein said tagged oligonucleotide further comprises a third region containing an RNA polymerase promoter, which third region is located 5 'to the second region.

39. The method of paragraph 37, wherein the 5'-end of this tag closing sequence is joined to the 5'-end of said tagged oligonucleotide.

40. The method of paragraph 39, wherein said tag closing sequence is modified to prevent DNA synthesis from starting therefrom.

41. The method of paragraph 40, wherein the 3'-base from this target hybridizing sequence is hybridized to the 5'-base from this tag closing sequence.

42. The method of paragraph 36, wherein the 3'-end of this tag closing sequence is joined to the 5'-end of said tagged oligonucleotide.

43. The method of paragraph 29, wherein in step (b) the target hybridizing sequence is hybridized to a tag closing oligonucleotide, said tagged oligonucleotide and said tag closing oligonucleotide being separate molecules.

44. The method of paragraph 43, wherein said tag closing oligonucleotide is modified to prevent DNA synthesis from starting.

45. The method of paragraph 44, wherein the 3'-terminal base of this target hybridizing sequence is hybridized to the 5'-terminal base of this tag closing oligonucleotide.

46. The method of paragraph 44, wherein both said tagged oligonucleotide and tag closing oligonucleotide are present in the nucleic acid sample during step (a) and wherein in step (a) this target hybridizing sequence favors hybridization with that target sequence nucleic acid, not with the tag closing oligonucleotide.

47. The method of paragraph 26, wherein the conditions of this nucleic acid amplification reaction are isothermal.

48. The method of paragraph 47, wherein said nucleic acid amplification reaction is a transcription based amplification reaction.

49. The method of paragraph 48, wherein said transcription based amplification reaction

107 is TMA.

50. The method of paragraph 48, wherein said first oligonucleotide comprises an RNA polymerase promoter that is located 5 'to this hybridizing sequence.

51. The method of paragraph 48, wherein said second oligonucleotide comprises a promoter for RNA polymerase that is located 5 'to this hybridization sequence, and wherein said tagged oligonucleotide further comprises a promoter for DNA polymerase that is located 5' from the other region.

52. The method of paragraph 26, wherein said target nucleic acid sequence is contained in that nucleic acid of a single species of microorganisms.

53. The method of paragraph 26, wherein said target nucleic acid sequence is contained in that nucleic acid of many species of microorganisms.

54. The method of paragraph 26, which method is selective for the amplification of a target nucleic acid sequence contained in each of a plurality of target nucleic acids and wherein in step (a) said target hybridizing sequence hybridizes to the 3'-end of the target nucleic acid sequence from each of these numerous target nucleic acids present in this nucleic acid sample.

55. The method of paragraph 54, wherein said target nucleic acid sequence contained in each of these multiple target nucleic acids is the same nucleic acid sequence.

56. The method of paragraph 54, wherein in step (c) this first oligonucleotide hybridizes to the 3'-terminus of each of the many target nucleic acid sequences present in this nucleic acid sample.

57. The method of paragraph 54, wherein the method comprises a plurality of first oligonucleotides, wherein in step (c) each of said plurality of first oligonucleotides hybridizes to the 3'end of the complement of at least one, but not all, of these numerous target nucleic acid sequences present in this nucleic acid sample.

58. The method of paragraph 54, wherein said tagged oligonucleotide is a universal bacterial oligonucleotide.

59. The method of paragraph 54, wherein said tagged oligonucleotide is a universal fungal oligonucleotide.

60. The method of paragraph 54, which method is a sterility test.

61. The method of paragraph 60, which method is a method of diagnosing sepsis.

108

62. The method of paragraph 61, wherein in step (a) said target hybridizing sequence hybridizes to the 3'-end of said target nucleic acid from each of those numerous target nucleic acids present in that nucleic acid sample, wherein said multiple target nucleic acids belong to the class of microorganisms selected from the group consisting of specific bacteria, Gram-positive bacteria, Gram-negative bacteria and fungi.

63. The method of paragraph 61, wherein each of these numerous target nucleic acids is a ribosomal nucleic acid.

64. The method of paragraph 26, wherein after step (b) this nucleic acid sample is exposed to a known contaminant source of that target nucleic acid sequence and wherein the production of these amplification products is essentially limited to the amplification of the target nucleic acid sequence provided in that nucleic acid sample, and not from this contaminating source of this target nucleic acid sequence.

65. The method of paragraph 64, wherein one or more of the components used in this method is a known contaminant source for this target nucleic acid.

66. The method of paragraph 65, wherein one or more of the reagents used in this amplification reaction are made from a material known to be a contaminating source for this target nucleic acid sequence.

67. The method of paragraph 66, wherein one or more of these reagents are prepared using a microorganism containing that target nucleic acid sequence.

68. The method of paragraph 64, wherein the environmental conditions of the method include a known contaminating source for this target nucleic acid sequence.

69. The method of paragraph 26, wherein at least a portion of this nucleic acid sample is obtained from a clinical, water, industrial, environmental, seed, beverage or food source.

70. The method of paragraph 26, wherein said target nucleic acid sequence is the target RNA sequence and wherein step (c) comprises:

extending this labeled oligonucleotide hybridized to this target nucleic acid sequence in a primer extension reaction using DNA polymerase to produce a first primer extension product containing a region complementary to this target nucleic acid sequence; separating this first primer extension product from this target nucleic acid sequence using an enzyme that selectively degrades that portion of that target nucleic acid that is hybridized with the first primer extension product;

treating the first primer extension product with the first oligonucleotide, which first oligonucleotide is a promoter oligonucleotide comprising a first and second region, said first region comprising a hybridizing sequence that hybridizes with that region of the first primer extension product, which is complementary to the 5'-end of this target nucleic acid sequence to form a hybrid promoter oligonucleotide: a first primer extension product, and the second region comprises a promoter for an RNA polymerase that is located 5 'to this first region; transcription from this hybrid promoter oligonucleotide: the first primer extension product of multiple copies of the first RNA product complementary to at least a portion of this first primer extension product using KNA polymerase which recognizes this promoter and begins transcription therefrom, the base sequence of the first the RNA product is substantially identical to the base sequence of this target nucleic acid sequence and the complement of this tag sequence;

treating the first RNA product with that second oligonucleotide, which to the second oligonucleotide is a priming oligonucleotide that hybridizes to the complement of this tag sequence to form a hybrid oligonucleotide primer: first RNA product, so that the primer extension reaction can be started from this priming oligonucleotide;

extending this priming oligonucleotide in a primer extension reaction using DNA polymerase to form a second primer extension product complementary to the first RNA product, which second primer extension product has a 3 'end that is complementary to the 5' end of the first RNA product;

separating this second primer extension product from this first RNA product using an enzyme that selectively degrades the first product

RNA;

treating the second primer extension product with this promoter oligonucleotide to form a hybrid promoter oligonucleotide: second primer extension product;

110 extending the 3'-end of this second primer extension product from this hybrid promoter oligonucleotide: a second primer extension product to attach a sequence complementary to that second region of this promoter oligonucleotide; and transcription from this hybrid promoter oligonucleotide: a second primer extension product of multiple copies of a second RNA product complementary to this second primer extension product using RNA polymerase, wherein the base sequence of this second RNA product is substantially identical to the base sequence of this target nucleic acid sequence and complete this tag sequence.

71. The method of paragraph 70, wherein step (a) further comprises treating said nucleic acid sample with a binding molecule that binds to said target nucleic acid sequence adjacent to or near the 5'-end of said nucleic acid sequence, and wherein the first product primer extension has a 3'-end that is determined by this binding molecule and which is complementary to the 5'-end of this target nucleic acid sequence.

72. The method of paragraph 71, wherein step (c) further comprises extending the 3 'end of this first primer extension product from said hybrid promoter oligonucleotide: first primer extension product to attach a complementary sequence to this promoter.

73. The method of paragraph 70, wherein said promoter oligonucleotide is modified to prevent DNA synthesis from starting.

74. The method of paragraph 70, wherein step (c) further comprises: extending said promoter oligonucleotide hybridized to said first primer extension product by DNA polymerase to produce a primer extension product complementary to that first primer extension product; and extending this promoter oligonucleotide hybridized to the second primer extension product to produce a primer extension product complementary to the second primer extension product.

75. The method of paragraph 70, wherein these separation steps are carried out utilizing the ribonuclease activity provided by this DNA polymerase.

76. The method of paragraph 70, wherein these separation steps are performed using ribonuclease activity provided by an enzyme other than this DNA polymerase.

77. The method of paragraph 26, wherein said target nucleic acid sequence is the target RNA sequence, and wherein step (c) comprises:

111 extending this tagged oligonucleotide hybridized to this target nucleic acid sequence in a primer extension reaction using DNA polymerase to form a first primer extension product containing a region complementary to that target nucleic acid sequence, said tagged oligonucleotide further comprising a third region located 5 'from the second region, which third region comprises a promoter for RNA polymerase;

separating this first primer extension product from this target nucleic acid sequence using an enzyme that selectively degrades that portion of that target nucleic acid that is hybridized with the first primer extension product;

treating this first primer extension product with the first oligonucleotide, which first oligonucleotide is a priming oligonucleotide that hybridizes to a region of the first primer extension product that is complementary to the 5'-end of this target nucleic acid sequence to form a hybrid oligonucleotide: first product primer extension, such that the primer extension reaction can start with this priming oligonucleotide;

extending this primer oligonucleotide in a primer extension reaction using DNA polymerase to form a second primer extension product complementary to the first primer extension product; and using this second primer extension product as a template for transcribing multiple copies of the first RNA product complementary to at least a portion of the second primer extension product using RNA polymerase that recognizes this promoter and starts transcribing therefrom, the base sequence of the first RNA product being substantially identical to the base sequence of this tag sequence and the complement of this target nucleic acid sequence;

78. The method of paragraph 77, wherein step (c) further comprises: treating said first RNA product with this priming oligonucleotide to form a hybrid priming oligonucleotide: first RNA product, such that the primer extension reaction can be started from this priming oligonucleotide;

extending this priming oligonucleotide in a primer extension reaction using DNA polymerase to form a third primer extension product complementary to the first RNA product, which is the third primer extension product

112 has a 3'-end that is complementary to the 5'-end of this first RNA product; separating this third primer extension product from the first RNA product using an enzyme that selectively degrades the first RNA product; treating this third primer extension product with the second oligonucleotide, which second oligonucleotide is a promoter oligonucleotide comprising the first and second regions, the first region comprising a hybridizing sequence that hybridizes to complement that tag sequence to create a promoter oligonucleotide hybrid: the third extension product a primer, such that the primer extension reaction can be started from this promoter oligonucleotide, and the second region comprises a promoter for RNA polymerase located 5 'to this first region;

extending this promoter oligonucleotide by primer extension reaction with this DNA polymerase to form a fourth primer extension product complementary to this third primer extension product;

extending this third primer extension product to add a sequence complementary to this promoter;

transcription from this hybrid promoter oligonucleotide: a third primer extension product of multiple copies of a second RNA product complementary to this third primer extension product using an RNA polymerase that recognizes this promoter and starts transcribing therefrom, the base sequence of this second RNA product being essentially identical to the base sequence of this tag sequence and the complement of this target nucleic acid sequence.

79. The method of paragraph 78, wherein these separation steps are performed utilizing the ribonuclease activity provided by this DNA polymerase.

80. The method of paragraph 78, wherein these separation steps are performed using ribonuclease activity provided by an enzyme other than this DNA polymerase.

81. The method of paragraph 26, wherein said target nucleic acid sequence is the target DNA sequence and wherein step (c) comprises:

extending this tagged oligonucleotide hybridized to this target nucleic acid sequence in a primer extension reaction using DNA polymerase to form a first primer extension product containing a region complementary to that target nucleic acid sequence; treating the first primer extension product with the first oligonucleotide, which first oligonucleotide is a promoter oligonucleotide comprising the first and second regions, said first region comprising a hybridization sequence that hybridizes with a region of the first primer extension product that is complementary to 5'-end of this target nucleic acid sequence to form a hybrid promoter oligonucleotide: the first primer extension product, and this second region is a promoter for RNA polymerase situated 5 'to this first region;

transcription from this hybrid promoter oligonucleotide: the first primer extension product of multiple copies of the first RNA product complementary to at least a portion of this first primer extension product using RNA polymerase that recognizes this promoter and begins transcription therefrom, the base sequence of the first RNA product being substantially identical to the base sequence of this target nucleic acid sequence and the complement of this tag sequence;

treating this first RNA product with that second oligonucleotide, which second oligonucleotide is a priming oligonucleotide that hybridizes to the complement of this tag sequence to form a hybrid oligonucleotide primer: first RNA product, such that the primer extension reaction can be started from this priming oligonucleotide;

extending this priming oligonucleotide in a primer extension reaction using DNA polymerase to form a second primer extension product comprising the complement of the first RNA product, which second primer extension product has a 3'-end that is complementary to the 5'-end of this first RNA product;

separating the second primer extension product from the first RNA product using an enzyme that selectively degrades the first RNA product;

treating the second primer extension product with this promoter oligonucleotide to form a hybrid promoter oligonucleotide: second primer extension product;

extending the 3'-end of this second primer extension product in this hybrid promoter oligonucleotide: second primer extension product to attach a sequence complementary to this promoter; and transcription from this hybrid promoter oligonucleotide: a second primer extension product of multiple copies of a second RNA product complementary to this second primer extension product using this RNA polymerase, wherein the base sequence of this second RNA product is substantially identical to the base sequence

114 this target nucleic acid sequence and the complement of this tag sequence.

82. The method of paragraph 81, wherein said promoter oligonucleotide is modified to prevent DNA synthesis from beginning.

83. The method of paragraph 81, wherein step (a) further comprises:

treatment of this nucleic acid sample with a displacement oligonucleotide that hybridizes to that target nucleic acid upstream of the tagged oligonucleotide such that a primer extension reaction can be started from this displacer oligonucleotide; and extending this displacer oligonucleotide in a primer extension reaction using DNA polymerase to form a third primer extension product that displaces the first primer extension product from the target nucleic acid.

84. The method of paragraph 81, wherein step (a) further comprises treating said nucleic acid sample with a binding molecule that binds to said target nucleic acid adjacent to or near the 5'-end of said target nucleic acid sequence, wherein the first product primer extension has a 3'-end that is determined by this binding molecule and which is complementary to the 5'-end of this target nucleic acid sequence.

85. The method of paragraph 84, wherein step (c) further comprises extending the 3'end of this first primer extension product in said hybrid promoter oligonucleotide: first primer extension product to attach a complementary sequence to this promoter sequence.

86. The method of paragraph 81, wherein step (c) further comprises:

extending this promoter oligonucleotide hybridized to this first primer extension product by DNA polymerase to produce a primer extension product complementary to this first primer extension product; and extending this promoter oligonucleotide hybridized to the second primer extension product by DNA polymerase to produce a primer extension product complementary to the second primer extension product;

87. The method of paragraph 81, wherein these separation steps are carried out utilizing the ribonuclease activity provided by this DNA polymerase.

88. The method of paragraph 81, wherein these separation steps are performed utilizing ribonuclease activity provided by an enzyme other than this DNA polymerase.

89. A kit for use in the selective amplification of at least one target nucleic acid sequence from a nucleic acid sample, which kit comprises: a tagged oligonucleotide comprising:

a first region comprising a target hybridizing sequence that hybridizes to the 3'-end of the target nucleic acid sequence under a first set of conditions, such that the first region can be extended in a matrix dependent manner in the presence of DNA polymerase; and a second region comprising the tag sequence situated 5 'to the first region, which second region does not stably hybridize to the target nucleic acid comprising that target nucleic acid sequence under the first set of conditions;

a tag closing sequence that hybridizes to that hybridizing sequence with a target under a second set of conditions, thereby blocking the hybridization of this tagged oligonucleotide with that target nucleic acid sequence, which tag closing sequence does not hybridize stably with that sequence with the target in the first set of conditions; and a first priming oligonucleotide that hybridizes to the complement of this tag sequence under this second set of conditions, such that the first priming oligonucleotide can be extended in a matrix dependent manner in the presence of DNA polymerase.

90. The kit of paragraph 89, wherein said tagged oligonucleotide further comprises a third region comprising an RNA polymerase promoter, which third region is located 5 'to the second region.

91. The kit of paragraph 89, wherein the 3'-base of this target hybridizing sequence hybridizes to the 5'-base of this tag closing sequence when said target hybridizing sequence is not hybridized to that target nucleic acid sequence under the second set of conditions.

92. The kit of paragraph 89, wherein the 5'-end of this tag closing sequence comprises a duplex stabilization moiety formed between that tag closing sequence and that target hybridizing sequence when said target hybridizing sequence is not hybridized to that target the nucleic acid sequence in the second set of conditions.

93. The kit of paragraph 89, wherein said tagged oligonucleotide and this tag closing sequence are separate molecules, said closing sequence

116 the tag is an tag closing oligonucleotide.

94. The kit of paragraph 89, wherein said tagged oligonucleotide and said tag closing sequence are contained in the same molecule.

95. The kit of paragraph 94, wherein said tag closing sequence is joined to said tagged oligonucleotide via a non-nucleotide linker.

96. The kit of paragraph 95, wherein said non-nucleotide linker contains at least one baseless nucleotide and polyethylene glycol.

97. The kit of paragraph 95, wherein the 3'-end of this tag closing sequence is joined to the 5'-end of this tagged oligonucleotide.

98. The kit of paragraph 95, wherein the 5'-end of this tag closing sequence is joined to the 5'-end of said tagged oligonucleotide.

99. The kit of paragraph 98, wherein said tag closing sequence hybridizes to that target hybridizing sequence to form an anti-parallel duplex when said target hybridizing sequence is not hybridized to that target nucleic acid sequence under that second set of conditions.

100. The kit of paragraph 99, wherein said tag closing sequence is modified to prevent DNA synthesis from starting therefrom.

101. The kit of paragraph 100, wherein said tag closing sequence is modified to include a blocking moiety located at its 3'-terminus. 101. The kit of paragraph 98, wherein said tag closing sequence hybridizes to that target hybridizing sequence to form a parallel duplex when this target hybridizing sequence is not hybridized to that target nucleic acid sequence under that second set of conditions.

103. The kit of paragraph 101, wherein said duplex comprises the 3'-terminal base of this target hybridizing sequence hybridized to the 3'-terminal base of this tag closing sequence.

104. The kit of paragraph 102, wherein said tag closing sequence is modified to prevent DNA synthesis from starting therefrom.

105. The kit of paragraph 104, wherein said tag closing sequence is modified to include a blocking moiety located at its 3'-terminus.

106. The kit of paragraph 89, wherein said first priming oligonucleotide stably hybridizes to this target nucleic acid sequence, and thus participates in the detectable amplification of this target nucleic acid sequence under the second set of conditions.

117

107. The kit of paragraph 89 further comprising a second priming oligonucleotide that hybridizes to the 5'-complement of this target nucleic acid sequence under this second set of conditions, such that the second satrter oligonucleotide can be extended in a matrix dependent manner in the presence of DNA polymerase.

108. The kit of paragraph 89 further comprising a promoter oligonucleotide comprising the first and second regions, said first region comprising a hybridizing sequence that hybridizes to the 5'-complement of this target nucleic acid sequence in the second set of conditions, and the second region comprising a polymerase promoter RNA that is located 5 'from this first region.

109. The kit of paragraph 108, wherein said promoter oligonucleotide is modified to prevent DNA synthesis from beginning.

110. The kit of paragraph 109, wherein said promoter oligonucleotide is modified to contain a blocking moiety located at its 3'-terminus.

111. The kit of paragraph 108, wherein said promoter oligonucleotide can be extended in a matrix-dependent manner in the presence of DNA polymerase when this hybridizing sequence is hybridized to the 5'-complement of this target nucleic acid sequence under this second set of conditions.

112. The kit of paragraph 89, further comprising DNA polymerase.

113. The kit of paragraph 112, wherein said DNA polymerase is reverse transcriptase.

114. The kit of paragraph 112, further comprising RNA polymerase.

115. Kit according to paragraph 89, further comprising nucleoside triphosphates.

116. The kit of paragraph 89, wherein said tagged oligonucleotide is free in solution.

117. The kit of paragraph 89, further comprising a solid support for binding the complex comprising this target nucleic acid and this tagged oligonucleotide.

118. The kit of paragraph 89, which kit does not contain a restriction enzyme capable of cleaving the duplex formed between this tag closing sequence and this target hybridizing sequence in the second set of conditions.

119. The kit of paragraph 89, wherein said target hybridizing sequence hybridizes to the 3'-end of multiple target nucleic acids under this first set of conditions.

120. The kit of paragraph 118, wherein said tagged oligonucleotide is a universal bacterial oligonucleotide.

118

121. The kit of paragraph 119, wherein said target hybridizing sequence hybridizes to a target region at the 3'-end of one or more target nucleic acid sequences, which target region is present in a number of microorganisms belonging to the class of microorganisms selected from the group consisting of specific bacteria , Gram-positive bacteria and Gram-negative bacteria in this first set of conditions.

122. The kit of paragraph 121, wherein said one or more target nucleic acid sequences are ribosomal nucleic acid sequences.

123. The kit of paragraph 119, wherein said tagged oligonucleotide is a universal fungal oligonucleotide.

124. The kit of paragraph 123, wherein said target hybridizing sequence hybridizes to a target region at the 3'-end of one or more target nucleic acid sequences, which target region is present in a number of fungal microorganisms, in the first set of conditions.

125. The kit of paragraph 124, wherein said one or more target nucleic acid sequences are ribosomal nucleic acid sequences

126. The pre-amplification reaction mixture for the selective amplification of one or more target nucleic acid sequences, which reaction mixture comprises:

a tagged oligonucleotide comprising the first and second regions, said first region comprising a target hybridizing sequence hybridized to a target region contained at the 3'-end of one or more target nucleic acid sequences present in this reaction mixture, and the second region comprises a tag sequence located 5 'to that of the target hybridizing sequence;

a first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-terminus of the complement of one or more of these target nucleic acid sequences; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of that tag sequence, which reaction mixture is substantially free of the active form of that tagged oligonucleotide that is not hybridized to that region contained in that one or more target nucleic acid sequences present in this reaction mixture, wherein the active form of this tagged oligonucleotide has an available hybridizing sequence with a target for hybridization with that target region present in the non-target nucleic acid added to this reaction mixture, and which reaction mixture does not contain a nucleic acid polymerase capable of extending any of these oligonucleotides in matrix-dependent method.

127. The reaction mixture of paragraph 126, wherein said tagged oligonucleotide contains an RNA polymerase promoter located 5 'to this tag sequence.

128. The reaction mixture of paragraph 126, wherein said tagged oligonucleotide does not contain an RNA polymerase promoter.

129. The reaction mixture according to paragraph 128, wherein said first oligonucleotide contains an RNA polymerase promoter located 5 'to this hybridizing sequence.

130. The reaction mixture of paragraph 129, wherein said first oligonucleotide has a blocking moiety situated at its 3'-end.

131. The reaction mixture of paragraph 129, wherein said target hybridizing sequence from this tagged oligonucleotide consists of SEQ ID NO: 19, its corresponding RNA, or complement thereof, and wherein said hybridizing sequence from this first oligonucleotide consists of SEQ ID NO : 20, the corresponding RJMA or their complement.

132. The reaction mixture of paragraph 126, wherein said first oligonucleotide contains an RNA polymerase promoter located 5 'to this hybridization sequence.

133. The reaction mixture of paragraph 132, wherein said first oligonucleotide has a blocking moiety situated at its 3'-terminus.

134. The reaction mixture of paragraph 126, wherein said tagged oligonucleotide comprises a tag closing sequence attached to its 5'-terminus.

135. The reaction mixture of paragraph 134, wherein said tagged oligonucleotide contains an RNA polymerase promoter located 5 'to this tag sequence.

136. The reaction mixture of paragraph 134, wherein said tagged oligonucleotide does not contain an RNA polymerase promoter.

137. The reaction mixture of paragraph 134, further comprising a free tagged oligonucleotide not hybridized to that target region of one or more target nucleic acid sequences, which free tagged oligonucleotide is in an inactive form that blocks or prevents hybridization of that free tagged oligonucleotide with complementary nucleic acids.

138. The reaction mixture according to paragraph 134, wherein the reaction mixture essentially does not contain an unhybridized form of this labeled oligonucleotide.

139. The reaction mixture according to paragraph 126, wherein the reaction mixture essentially does not contain an unhybridized form of this labeled oligonucleotide.

140. The reaction mixture of paragraph 126, wherein said tagged oligonucleotide does not contain a tag closing sequence and which reaction mixture does not contain a tag closing oligonucleotide.

141. The reaction mixture of paragraph 126, further comprising a probe for detecting one or more amplification products comprising the one or more target nucleic acid sequences or their complement.

142. The reaction mixture of paragraph 126, wherein said inactive form of this tagged oligonucleotide comprises a tag closing sequence hybridized to that target hybridizing sequence.

143. A reaction mixture for amplifying a target nucleic acid sequence, which reaction mixture comprises: a tagged oligonucleotide comprising the first and second regions, said first region comprising a target hybridizing sequence hybridized to the 3'-end of the target nucleic acid sequence and the second region comprises a tag sequence 5 'to that of the target hybridizing sequence; a first oligonucleotide comprising a hybridizing sequence that hybridizes to the 3'-terminus of the complement of this target nucleic acid sequence; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of this tag sequence, wherein substantially all of the unhybridized labeled oligonucleotide in this reaction mixture is inactive, which blocks or prevents the hybridization of this unhybridized nucleated acid with that oligonucleotide .

144. The reaction mixture of paragraph 143, wherein said tagged oligonucleotide and said tag closing sequence are separate molecules, said tag closing sequence being a tag closing oligonucleotide.

145. The reaction mixture of paragraph 143, wherein said tagged oligonucleotide and this tag closing sequence are contained in the same molecule.

146. The reaction mixture of paragraph 143, wherein said tagged oligonucleotide is not bound to a solid support.

121

LIST OF SEQUENCES [0286] &lt;110&gt; GEN-PROBE INCORPORATED BECKER, Michael M.

LIVEZEY, Kristin W.

LAM, Wai-Chung W.

&lt;120&gt; MARKED OLIGONUCLEOTIDES AND THEIR APPLICATION FOR NUCLEIC ACID AMPLIFICATION &lt;130&gt; GP193-PCT &lt;140&gt; will be assigned &lt;141&gt; 2007-06-06 &lt;150&gt; 60/871442 &lt;151&gt; 2006-12-21 &lt;150&gt; 60 / 811 581 15 &lt;151&gt; 2006-06-06 &lt;160&gt; 24 &lt;170&gt; patentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 60 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; labeled HCV-specific priming oligonucleotide &lt;400&gt; 1 gtttgtatgt ctgttgctat tatgtctaca ggcattgagc gggttgatcc aagaaaggac 60 &lt;210&gt; 2 &lt;211&gt; 23 &lt;212&gt; DNA

122 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; tag specific priming oligonucleotide &lt;400&gt; 2 gtttgtatgt ctgttgctat tat 23 &lt;210&gt; 3 &lt;211&gt; 60 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; HCV-specific promoter oligonucleotide &lt;400&gt; 3 atttaatacg actcactata gggagaccac aacggtttct agccatggcg ttagtatgag &lt;210&gt; 4 &lt;211&gt; 26 &lt;212&gt; RNA &lt;213&gt; Hepatitis C virus &lt;220&gt;

&lt;221&gt; misc_feature 20 &lt;222&gt; (1) .. (26) &lt;223&gt; 2'-methoxy analogues &lt;400&gt; 4 auggcuagac gcuuucugcg ugaaga 26 &lt;210&gt; 5 25 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial &lt; 220&gt;

123 &lt;223&gt; extension oligonucleotide &lt;400&gt; 5 tgtcgtgcag crtccaggac cccccctccc gggagagcca ta 42 &lt;210&gt; 6 5 &lt;211&gt; 52 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; HCV-specific capture probe &lt;400&gt; 6 gggcacucgc aagcacccut ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 52 &lt;210&gt; 7 &lt;211&gt; 51 &lt;212&gt; DNA &lt;213&gt;

&lt;223&gt; HCV-specific capture probe &lt;400&gt; 7 cauggugcac ggucuacgtt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 51 &lt;210&gt; 8 &lt;211&gt; 23 &lt;212&gt; RNA &lt;220&gt; Artificial &lt;220&gt;

&lt;223&gt; HCV-specific molecular torch hybridization probe &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (23)

124 &lt;223&gt; 2'-methoxy analogues &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (18) .. (19) &lt;223&gt; non-nucleotide linker &lt;400&gt; 8 cguuccgcag accacuauga acg 23 &lt;210&gt; 9 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Escherichia coli &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (4) &lt;223&gt; 2'-methoxy analogues &lt;400&gt; 9 cugctggcac ggagttagcc ggtgcttc 28 &lt;210&gt; 10 &lt;211&gt; 47 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; E. coli specific promoter oligonucleotide &lt;400&gt; 10 atttaatacg actcactata gggagagaag gccttcgggt tgtaaag 47 &lt;210&gt; 11 &lt;211&gt; 18 &lt;212&gt; RNA &lt;213&gt; Escherichia coli

125 &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (18) &lt;223&gt; 2'-methoxy analogues &lt;400&gt; 11 gccuucuuca uacacgcg 18 &lt;210&gt; 12 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; molecular torch hybridization probe specific for E. coli &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (24) &lt;223&gt; 2'-methoxy analogues &lt;220&gt;

&lt;221&gt; misc_feature 5 &lt;222&gt; (24) .. (25) &lt;223&gt; non-nucleotide linker &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (25) .. (29) &lt;223&gt; DNA &lt;400&gt; 12 cugcggguaa cgucaaugag caaacgcag 29 &lt;210&gt; 13 &lt;211&gt; 1542 &lt;212&gt; DNA

126 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic E. rRNA template coli &lt;400&gt; 13 aaattgaaga gtttgatcat ggctcągatt gaacgctggc ggcaggccta acacatgcaa 60 gtegaacggt aaćaggaaga agcttgcttc tttgctgacg agtggcggac gggtgagtaa 120 tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct aataccgcat 180 aaegtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg tgcccagatg 240 ggattagcta gtaggtgggg taacggctca cctaggcgac gatccctagc tggtctgaga 300 ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag gcageagtgg 360 ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg aagaaggcct 420 tcgggttgta aagtaćtttc agcggggagg aagggagtaa ągttaatacc tttgctcatt 480 gacgttaccc gcagaagaag caccggctaa ccccgtgcca gcagccgcgg taatacggag 540 ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt tgttaagtca 600 gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag cttgagtctc 660 gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 720 ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca 780 aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc 840 cttgaggcgt ggcttccgga gctaacgcgt taagtcgacc gcctggggag tacggccgca 900 aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960 tcgatgcaac gcgaagaacc ttacctggtc ttgacatcca cggaagtttt cagagatgag 1020 aatgtgcctt cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga 1080 aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc 1140 cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc 1200 atcatggccc ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg 1260 acctcgcgag agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac 1320 tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt 1440 agcttaacct tcgggagggc gcttaccact ttgtgattca tgactggggt gaagtcgtaa 1500 caaggtaacc gtaggggaac ctgcggttgg atcacctcct the 1342 &lt;210&gt; 14 &lt;211&gt; 57 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

127 &lt;223&gt; labeled primer oligonucleotide specific for E. coli &lt;400&gt; 14 gtttgtatgt ctgttgctat tatgtctacc tgctggcacg gagttagccg gtgcttc 57 &lt;210&gt; 15 5 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; tag specific priming oligonucleotide &lt;400&gt; 15 gtttgtatgt ctgttgctat tat 23 &lt;210&gt; 16 &lt;211&gt; 51 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; non-specific capture probe &lt;220&gt;

&lt;221&gt; misc-feature 20 &lt;222&gt; (1) .. (18) &lt;223&gt; 2'-methoxy analogues &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (19) .. (51) &lt;223&gt; DNA &lt;400&gt; 16 kkkkkkkkkkkkkkkkkktt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 51 &lt;210&gt; 17

128 &lt;211&gt; 22 &lt;212&gt; RNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; molecular torch hybridization probe specific for E. coli &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (22) &lt;223&gt; 2'-methoxy analogues &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (17) .. (18) &lt;223&gt; non-nucleotide linker &lt;400&gt; 17 cgagcaaagg uauuaacgcu cg 22 &lt;210&gt; 18 &lt;211&gt; 29 &lt;212&gt; RNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; molecular torch hybridization probe specific for E. coli &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (1) .. (29) &lt;223&gt; 2'-methoxy analogues &lt;220&gt;

&lt;221&gt; misc_feature &lt;222&gt; (24) .. (25) &lt;223&gt; non-nucleotide linker

129 &lt;400&gt; 18 cgagcaaagg uauuaacuuu acucgcucg &lt;210&gt; 19 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Escherichia coli &lt;400&gt; 19 gtctacctgc tggcacggag ttagccggtg cttc 34 &lt;210&gt; 20 &lt;211&gt; 21 &lt;212&gt; DNA &lt;213&gt; &lt;400&gt; 20 gaaggccttc gggttgtaaa g 21 &lt;210&gt; 21 &lt;211&gt; 23 &lt;212&gt; RNA &lt;213&gt; Escherichia coli &lt;400&gt; 21 ugcggguaac gucaaugagc aaa 23 &lt;210&gt; 22 &lt;211&gt; 16 &lt;212&gt; RNA &lt;213&gt; Escherichia coli &lt;400&gt; 22 gagcaaaggu auuaac 16 &lt;210&gt; 23 &lt;211&gt; 23 &lt;212&gt; RNA

130 &lt;213&gt; Escherichia coli &lt;400&gt; 23 gagcaaaggu auuaacuuua cuc 23 &lt;210&gt; 24 &lt;211&gt; 39 &lt;212&gt; DNA &lt;213&gt; Escherichia coli &lt;400&gt; 24 tgcgggtaac gtcaatgagc aaaggtatta actttactc 39