Tagged oliggonucleotides and their use in nucleic acid amplification methods

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DESCRIPTION

FIELD OF THE INVENTION

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 that may be present alone or as a component of a homogeneous or heterogeneous mixture of nucleic acids. The nucleic acid mixture can be that found in a sample taken for a diagnostic test, screening of blood products, sterility analysis, microbiological detection in food, water, beverages, industrial or environmental samples, research studies, preparation of reagents or materials for other procedures such as cloning, or for other purposes. Selective amplification of specific nucleic acid sequences, as described herein, is of particular value in any of a variety of detection assays to increase the accuracy and reliability of such assays, while at the same time reducing the preparation, purification and / or sterilization requirements for the reagents used in the assays, and for the medium in which the assays are carried out.

DESCRIPTION OF THE RELATED TECHNIQUE

Detection and / or quantification of specific nucleic acid sequences is an important technique for detecting and classifying microorganisms, for diagnosing infectious diseases, for measuring response to various types of treatments, and the like. Such procedures are also useful in the detection and quantification of microorganisms in food products, water, beverages, industrial and environmental samples, seed stocks, and other types of materials in which it may be necessary to monitor the presence of specific microorganisms.

Numerous amplification-based methods for the detection and quantification of target nucleic acids are well known and established in the art. The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, renaturation of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target sequence (eg. , Mullis et al., Process for Amplifying, Detecting and / or Cloning Nucleic Acid Sequences, US Pat. No. 4,683,195; Mullis, Process for Amplifying Nucleic Acid Sequences, US Pat. No. 4,683,202; Mullis et al., Process for Amplifying, Detecting and / or Cloning Nucleic Acid Sequences, pat. US No. 4,800,159; Gelfand et al., Reaction Mixtures for the Detection of Target Nucleic Acids, pat. US No. 5,804,375; Mullis et al. (1987) Meth. Enzymol. 155, 335-350; and Murakawa et al. (1988) DNA 7, 287-295).

In a variation called RT-PCR, reverse transcriptase (RT) is used to produce complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA (Gelfand et al., Reverse Transcription with Thermostable DNA Polymerases - High Temperature Reverse Transcription, US Pat. Nos. 5,322,770 and 5,310,652).

Another well-known amplification method is strand displacement amplification, commonly referred to as SDA, which uses cycles of renaturation of pairs of primer sequences to opposite strands of a target sequence, elongation of the primers in the presence of a dNTP to produce a double-stranded hemiphosphorothioate primer extension product, the formation of an endonuclease-mediated nick at a hemi-modified restriction endonuclease recognition site, and the polymerase-mediated extension of the primers from the 3 'end of the nick to displace the existing strand and produce a strand for the next round of primer renaturation, nicking, and strand displacement, resulting in geometric amplification of the product (eg, Walker, G. et al. (1992), Proc. Natl. Acad. Sci. USA 89, 392-396; Walker et al., Nucleic Acid Target Generation, pat. U.S. No. 5,270,184; Walker, Strand Displacement Amplification, pat. U.S. No. 5,455,166; and Walker et al. (1992) Nucleic Acids Research 20, 1691-1696). Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (European Pat. No. 0 684 315).

Other amplification methods include rolling circle amplification (RCA) (eg, Lizardi, Rolling Circle Replication Reporter Systems, US Pat. No. 5,854,033); helicase-dependent amplification (HDA) (eg, Kong et al., Helicase Dependent Amplification Nucleic Acids, US Patent Sol. Pub. No. Us 2004-0058378 A1); and loop-mediated isothermal amplification (LAMP) (eg, Notomi et al., Process for Synthesizing Nucleic Acid, US Pat. No. 6,410,278).

Transcription-based amplification methods commonly used in the art include nucleic acid sequence-based amplification, also referred to as NASBA (eg, Malek et al., US Pat. No. 5,130,238) ; methods that rely on the use of an RNA replicase to amplify the probe molecule itself, commonly referred to as Qp replicase (eg, Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202); transcription-based amplification methods (eg, Kwoh, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173-1177) and self-sustained sequence replication (eg. eg, Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878;

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Landgren (1993) Trends in Genetics 9, 199-202; and HELEN H. LEE et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)).

Another transcription-based amplification method is transcription-mediated amplification, commonly referred to as TMA, which synthesizes multiple copies of a target nucleic acid sequence autocatalytically under substantially constant conditions of temperature, ionic strength, and pH, in which multiple RNA copies of the target sequence generate additional copies autocatalytically (eg, Kacian et al., Nucleic Acid Sequence Amplification Methods, pat. U.S. No. 5,480,784; and Kacian et al., pat. U.S. No. 5,399,491). TMA is a robust and highly sensitive amplification system with proven efficiency, which overcomes many of the problems associated with PCR-based amplification systems. In particular, temperature cycling is not necessary.

Amplification assays are especially suitable for the detection of microorganisms in the context of clinical laboratory tests, bioprocess monitoring, or any other situation in which the detection of microorganisms in a particular type of sample is desired, offering sensitivity high and fast time to obtain results compared to conventional microbiological techniques. In addition, amplification methods can be used in the detection of the large number of microorganisms that are difficult or impossible to grow on synthetic media. However, there are certain limitations associated with first-generation amplification assays that have limited their acceptance in certain situations, such as clinical microbiology laboratories. An inherent problem associated with the high sensitivity of nucleic acid amplification systems is that contaminating nucleic acid introduced into the amplification system (e.g., from one or more reagents used during amplification, from the technician performing the assay, the medium in which the amplification is carried out, etc.) may give false positive results. For example, even extremely small amounts of nucleic acid contamination present in reagents and / or enzymes used in an amplification reaction, or in the medium in which the amplification reaction is carried out, can result in a positive amplification signal despite the fact that the sequence of interest is not present in the nucleic acid sample being tested. This requires significant effort to be invested in sample preparation, purification, sterilization, etc. of the reagents used in amplification reactions to avoid or minimize false positive results.

Therefore, there remains a need in the art for a robust nucleic acid amplification system that can selectively amplify one or more target nucleic acid sequences of interest while reducing or eliminating false positive results that may arise. as a result of contaminating biological material, such as contaminating nucleic acid. There also remains a need for amplification systems that have reduced reagent purification and / or sterility requirements. As further described herein, the present invention meets these needs and offers other related advantages.

SUMMARY OF THE INVENTION

The present invention is generally directed to nucleic acid amplification methods that desirably reduce or eliminate false positive amplification signals that result from contaminating biological material, eg, nucleic acid, that may be present in one or more reagents, components, or materials that are used in an amplification reaction, or that are present in the medium in which the amplification reaction is carried out. The invention further offers the advantage of requiring less stringent purification and / or sterility effort than is conventionally necessary to ensure that the enzymes and other reagents and components used in amplification reactions are free from contamination by bacterial and other nucleic acids. which can produce false positive results. Such components or materials include, but are not limited to, water, buffers, salts, solid supports (eg, magnetically charged particles or spheres), and receptacles (eg, glass or plastic objects). Therefore, the methods of the invention are useful for detecting and / or quantifying microorganisms in clinical samples, food products, water, industrial and environmental samples, seed stocks, and other types of materials in which it may be necessary to detect and / or or monitor the presence of microorganisms. The methods of the invention have particular advantages for the analysis of raw materials used in the production of products for the biotechnology, pharmacy, cosmetic and beverage industries, for the analysis before the commercialization of the final products, and for the screening of the sterility to test a class of organisms or the total viable organisms in a material of interest (fungal bacteria or both). In clinical situations, the methods of the invention would be especially useful for the analysis of sepsis, especially sepsis, which is caused by pathogenic organisms and / or their toxins in the bloodstream. The methods of the invention are defined in the appended claims.

According to one embodiment of the present disclosure, methods are provided for the selective amplification of at least one target nucleic acid sequence, such as a DNA sequence or an RNA sequence, wherein the method comprises the steps of: (a) treat an acid sequence

ES 2 358 296 T3 target nucleic acid in a nucleic acid sample, eg, in which the target nucleic acid is immobilized on a solid support, with a heterologous marker sequence to produce a labeled target nucleic acid sequence; (b) reducing in said sample the effective concentration of heterologous marker sequences that have not been part of said tagged target nucleic acid sequence and that are in a form capable of producing a target nucleic acid sequence tagged with said nucleic acid sequence target; and (c) subjecting said labeled target nucleic acid sequence to reagents and conditions sufficient for detectable amplification of the target nucleic acid sequence, wherein the submitting step exposes the nucleic acid sample to a known contaminating source of the target nucleic acid sequence after step (b), and wherein the detectable amplification of the target nucleic acid sequence is substantially limited to the amplification of the target nucleic acid sequence contributed by the labeled target nucleic acid sequence of step (a), and not by the acid sequence. nucleic target contributed by the known pollutant source.

The methods of the disclosure are particularly useful when one or more reagents or components used are produced with a material that is known to be a contaminating source of a target nucleic acid sequence to be amplified. In one example, one or more of the reagents used in the methods, such as nucleic acid polymerases, are produced using a microorganism that contains the target nucleic acid sequence. In another example, components used in the methods, such as reaction vessels, pipette tips, and solid supports for binding the labeled target nucleic acid sequences, can be a known contaminating source of the nucleic acid sequence. target. Furthermore, the methods are useful when the environmental conditions in which amplification is carried out include a known contaminating source of a target nucleic acid sequence, such as ambient air, the operator, or analytical instrumentation.

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

In another particular aspect, step (b) comprises diluting or eliminating the heterologous marker sequences that have not formed part of the marked target nucleic acid sequence of the nucleic acid sample. In an alternative aspect, step (b) comprises inactivating heterologous marker sequences that have not been part of said labeled target nucleic acid sequence to produce an inactivated heterologous marker sequence. In a related aspect, the method further comprises removing the inactivated heterologous marker sequence from said nucleic acid sample during step (b). The heterologous marker sequence can be inactivated by blocking its ability to complex with the target nucleic acid sequence, by using an enzyme to digest a component or cleave a site from a complexed portion of the heterologous marker sequence, chemically altering the heterologous marker sequence. , or by other means altering the ability of the heterologous marker sequence to complex with the target nucleic acid sequence in an amplification reaction mixture.

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

In yet another aspect, the heterologous marker sequence has an active form during step (a) that allows the heterologous marker sequence to produce the labeled target nucleic acid sequence, and wherein the heterologous marker sequence that has not produced the labeled target nucleic acid sequence is converted to an inactive form in step (b) that blocks the heterologous marker sequence from producing a labeled target nucleic acid sequence during step ( c).

The target hybridization sequence, in certain aspects, is a universal oligonucleotide, such as a universal bacterial or fungal oligonucleotide.

Step (c) comprises producing amplification products in a nucleic acid amplification reaction by using first and second oligonucleotides, and the first oligonucleotide comprises a sequence that hybridizes to a 3 'end of the complement of the target nucleic acid sequence. and the second oligonucleotide comprises a sequence that hybridizes to a complement of the marker sequence but 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 part of the marker sequence.

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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.

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

For example, in a particular aspect, the method is selective for the amplification of a target nucleic acid sequence contained in each of a variety of target nucleic acids, and wherein the heterologous marker sequence produces a labeled target nucleic acid sequence. with the target nucleic acid sequence of each of the variety of target nucleic acids present in the nucleic acid sample in step (a). 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.

In another particular aspect, the method is selective for the amplification of multiple bacterial or fungal target nucleic acid sequences, eg, where 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 members of a group of bacterial species including Staphylococci spp. (eg, Staphylococcus aureus, Staphylococcus epidermis, and Staphylococcus haemolyticus), Streptococci spp. (eg, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci, and beta-hemolytic streptococci), Enterococcus spp. (eg, Enterococcus faecium and Enterococcus faecalis), Escherichia spp. (eg, Escherichia coli), Klebsiella spp. (eg, Klebsiella pneumoniae and Klebsiella oxytoca), Pseudomonas spp. (eg, Pseudomonas aeruginosa), Enterobacter spp. (eg, Enterobacter cloacae and Enterobacter aerogenes), Proteus spp. (eg, Proteus mirabilis), Bacterioides spp., Clostridium spp., Serratia spp. (eg, Serratia marcescens), Acinetobacter spp. (eg, Acinetobacterbaumannii) and Stenotrophomonas spp. (eg, Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in a sepsis test.

In another aspect, the method is selective for the amplification of target nucleic acid sequences obtained from members of a group of fungal species including Candida spp. (eg, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis, and Candida famata), Histoplama capsulatum, Cryptococcus spp. (eg, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii) Coccidioides spp. (eg, Coccidioides immitis), Trichosporon spp. (eg, Trichosporon cutaneum), Malassezia spp. (eg, Malassezia furfur), Rhodotorula spp., Nocardia spp. (eg, Nocardia asteroides), Fusarium spp. and Asperigillus spp. (eg, Asperigillus fumigatus). At least a portion of these microorganisms would be suitable for detection in a sepsis test.

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

The methods are especially suitable, in certain respects, for use in the analysis of sterility or the diagnostic analysis of sepsis.

According to another embodiment of the disclosure, there is provided a method for the selective amplification of at least one target nucleic acid sequence from a nucleic acid sample, and the method comprises the steps of: (a) treating a nucleic acid sample comprising a target nucleic acid sequence with a labeled oligonucleotide comprising first and second regions, and the first region comprises a target hybridizing sequence that hybridizes to a 3 'end of the sequence of target nucleic acid and the second region comprises a marker sequence located 5 'to the target hybridization sequence, wherein the second region does not stably hybridize to a target nucleic acid containing the target nucleic acid sequence; (b) reducing in said nucleic acid sample the effective concentration of unhybridized labeled oligonucleotide having an active form in which a target hybridizing sequence of said unhybridized labeled oligonucleotide is available for hybridization to said target nucleic acid sequence ; and (c)

ES 2 358 296 T3 produce amplification products in a nucleic acid amplification reaction through the use of first and second oligonucleotides, in which the first oligonucleotide comprises a hybridization sequence that hybridizes to a 3 'end of the complement of the sequence of target nucleic acid and the second oligonucleotide comprises a hybridization sequence that hybridizes to the complement of the marker sequence, wherein the second oligonucleotide stably hybridizes to the target nucleic acid, 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 comprising a base sequence that is substantially identical or complementary to all or part of the marker sequence.

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

The effective concentration of the unhybridized labeled oligonucleotide in active form prior to amplification is preferably reduced by diluting the nucleic acid sample or inactivating and / or removing the unhybridized labeled oligonucleotide. In one aspect, step (b) comprises inactivating the unhybridized labeled oligonucleotide so that the unhybridized labeled oligonucleotide does not stably hybridize to the target nucleic acid sequence during step (c). In an example of inactivation, a labeled oligonucleotide has an active form during step (a) that allows the target hybridization sequence to hybridize to the target nucleic acid sequence, and in which the unhybridized labeled oligonucleotide is converted to a inactive form in step (b) that blocks or prevents the labeled oligonucleotide from hybridizing to the target nucleic acid sequence during step (c). The labeled oligonucleotide can be inactivated by blocking that the target hybridization sequence hybridizes to the target nucleic acid sequence, by using an enzyme to digest a component or to cleave a site on a double-stranded molecule formed between the hybridization sequence to the target and target nucleic acid sequence, chemically altering the hybridization sequence to the target, or by other means altering the ability of the labeled oligonucleotide to hybridize to the target nucleic acid sequence in an amplification reaction mixture.

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

In another example where step (b) comprises inactivating the unhybridized tagged oligonucleotide, the unhybridized tagged oligonucleotide from step (a) is converted from a single-stranded form to a double-stranded molecule form in step (b). The double-stranded molecule form may be a hairpin tag molecule comprising a tag closure sequence attached to a 5 'end of the tagged oligonucleotide, wherein the tag closure sequence hybridizes to the target hybridization sequence under the conditions from step (b), thereby blocking the hybridization of the unhybridized labeled oligonucleotide from step (a) to the target nucleic acid sequence in steps (b) and (c). In another aspect, the tag closure sequence is linked to the tagged oligonucleotide via a non-nucleotide spacer. For example, a 5 'end of the tag closure sequence may be linked to a 5' end of the tagged oligonucleotide.

The labeled oligonucleotide may further comprise a third region containing a promoter for an RNA polymerase, and the third region is located 5 'to the second region.

In another aspect, the tag closure sequence is modified to prevent initiation of DNA synthesis from it.

According to another aspect, a 3'-terminal base of the target hybridization sequence hybridizes to a 5'-terminal base of the tag closure sequence. In another aspect, a 3 'end of the tag closure sequence is joined to a 5' end of the tagged oligonucleotide.

In yet another aspect, the target hybridization sequence hybridizes to a tag closure oligonucleotide in step (b), and the tagged oligonucleotide and the tag closure oligonucleotide are separate molecules. The marker closure oligonucleotide can be modified, if desired, to prevent the initiation of DNA synthesis from it.

Furthermore, in certain aspects, a 3'-terminal base of the target hybridizing sequence hybridizes to a 5'-terminal base of the tag closure oligonucleotide.

In other aspects, the tagged oligonucleotide and the tag closure oligonucleotide are present in the nucleic acid sample during step (a), and wherein the target hybridization sequence favors hybridization to the target nucleic acid sequence on the marker closure oligonucleotide in step (a).

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As indicated above, the methods of the invention can employ any of a variety of amplification techniques. In certain cases, it may be preferred to use an isothermal amplification reaction, such as a transcription-based amplification reaction, preferably TMA or real-time TMA.

In a particular aspect, the first oligonucleotide comprises a promoter for an RNA polymerase that is located 5 'to the hybridization sequence. In another aspect, the second oligonucleotide comprises a promoter for an RNA polymerase that is located 5 'to the hybridization sequence, and wherein the labeled oligonucleotide further comprises a promoter for an RNA polymerase that is located 5' to the second region.

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

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

In a particular embodiment, the method is selective for the amplification of multiple bacterial or fungal target nucleic acid sequences, eg, where the 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 members of a group of bacterial species including Staphylococci spp. (eg, Staphylococcus aureus, Staphylococcus epidermis, and Staphylococcus haemolyticus), Streptococci spp. (eg, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci, and beta-hemolytic streptococci), Enterococcus spp. (eg, Enterococcus faecium and Enterococcus faecalis), Escherichia spp. (eg, Escherichia coli), Klebsiella spp. (eg, Klebsiella pneumoniae and Klebsiella oxytoca), Pseudomonas spp. (eg, Pseudomonas aeruginosa), Enterobacter spp. (eg, Enterobacter cloacae and Enterobacter aerogenes), Proteus spp. (eg, Proteus mirabilis), Bacterioides spp., Clostridium spp., Serratia spp. (eg, Serratia marcescens), Acinetobacter spp. (eg, Acinetobacterbaumannii) and Stenotrophomonas spp. (eg, Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in a sepsis test.

In another particular embodiment, the method is selective for the amplification of target nucleic acid sequences obtained from members of a group of fungal species that include Candida spp. (eg, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis, and Candida famata), Histoplama capsulatum, Cryptococcus spp. (eg, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii) Coccidioides spp. (eg, Coccidioides immitis), Trichosporon spp. (eg, Trichosporon cutaneum), Malassezia spp. (eg, Malassezia furfur), Rhodotorula spp., Nocardia spp. (eg, Nocardia asteroides), Fusarium spp. and Asperigillus spp. (eg, Asperigillus fumigatus). At least a portion of these microorganisms would be suitable for detection in a sepsis test.

In certain aspects, the target hybridization sequence hybridizes to a 3 'end of each of the multiple target nucleic acid sequences present in the nucleic acid sample in step (a). Furthermore, the first oligonucleotide hybridizes to a 3 'end of the complement of each of the multiple target nucleic acid sequences present in the nucleic acid sample in step (c).

The method may further comprise a plurality of first oligonucleotides, and each of the plurality of first oligonucleotides hybridizes to a 3 'end of the complement of at least one, but less of all, of the multiple target nucleic acid sequences present in the sample. nucleic acid in step (c).

The labeled oligonucleotide, in a particular embodiment of the invention, is a universal bacterial oligonucleotide or a universal fungal oligonucleotide. Such labeled oligonucleotides are

ES 2 358 296 T3 especially suitable for sterility analysis methods, such as methods for analyzing bioprocess materials or that are diagnostic of sepsis.

In a more particular aspect, the hybrid target hybridization sequence to a 3 'end of the target nucleic acid of each of the diversity of target nucleic acids present in the nucleic acid sample in step (a), and the diversity of Target nucleic acids belong to a class of microorganisms selected from the group consisting of Eubacteria, gram-positive bacteria, gram-negative bacteria, and fungi. In another aspect, each of the variety of target nucleic acids is a ribosomal nucleic acid.

In another aspect of the disclosure, the multiple target nucleic acid sequences include members belonging to a class of bacterial microorganisms selected from the group consisting of Staphylococci spp. (eg, Staphylococcus aureus, Staphylococcus epidermis, and Staphylococcus haemolyticus), Streptococci spp. (eg, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci, and beta-hemolytic streptococci), Enterococcus spp. (eg, Enterococcus faecium and Enterococcus faecalis), Escherichia spp. (eg, Escherichia coli), Klebsiella spp. (eg, Klebsiella pneumoniae and Klebsiella oxytoca), Pseudomonas spp. (eg, Pseudomonas aeruginosa), Enterobacter spp. (eg, Enterobacter cloacae and Enterobacter aerogenes), Proteus spp. (eg, Proteus mirabilis), Bacterioides spp., Clostridium spp., Serratia spp. (eg, Serratia marcescens), Acinetobacter spp. (eg, Acinetobacterbaumannii) and Stenotrophomonas spp. (eg, Stenotrophomonas maltophilia). At least a portion of these microorganisms would be suitable for detection in a sepsis test.

In a further aspect of the disclosure, the multiple target nucleic acid sequences include members belonging to a class of fungal microorganisms selected from the group consisting of Candida spp. (eg, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis, and Candida famata), Histoplama capsulatum, Cryptococcus spp. (eg, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii) Coccidioides spp. (eg, Coccidioides immitis), Trichosporon spp. (eg, Trichosporon cutaneum), Malassezia spp. (eg, Malassezia furfur), Rhodotorula spp., Nocardia spp. (eg, Nocardia asteroides), Fusarium spp. and Asperigillus spp. (eg, Asperigillus fumigatus). At least a portion of these microorganisms would be suitable for detection in a sepsis test.

The nucleic acid sample is often exposed to a known contaminating source of the target nucleic acid sequence after step (b), and therefore the described methods allow the production of the amplification products to be substantially limited to amplification of the target nucleic acid sequence contributed by the nucleic acid sample, and not by the contaminating source of the target nucleic acid sequence. For example, one or more reagents or components used in the amplification reaction is a known contaminating source of the target nucleic acid sequence. Alternatively, or additionally, one or more reagents are produced with a material known to be a contaminating source of the target nucleic acid sequence, such as nucleic acid polymerases produced using microorganisms known to contain the target nucleic acid sequence. Furthermore, the environmental conditions in which the method is carried out may include a known contaminating source of the target nucleic acid sequence. In a particular aspect, at least a part of said nucleic acid is obtained from a clinical, water, industrial, environmental, seed, beverage or food source.

According to another embodiment of the present disclosure, the target nucleic acid sequence is an RNA target sequence, and step (c) comprises: extending the labeled oligonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction with 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 by using an enzyme that selectively degrades that part of the target nucleic acid hybridized to the first primer extension product; treat the first primer extension product with the first oligonucleotide, and the first oligonucleotide is a promoter oligonucleotide comprising first and second regions, and the first region comprises a hybridization sequence that hybridizes to a region of the first primer extension product that is complementary to a 5 'end of the target nucleic acid sequence to form a promoter oligonucleotide hybrid: first primer extension product, and the second region comprises a promoter for an RNA polymerase that is located 5 'to the first region; transcribing from the promoter oligonucleotide hybrid: first primer extension product multiple copies of a first RNA product complementary to at least a part of the first primer extension product using an RNA polymerase that recognizes the promoter and initiates transcription from him, wherein the base sequence of the first RNA product is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the marker sequence; treat the first RNA product with the second oligonucleotide, and the second oligonucleotide is a primer oligonucleotide that hybridizes to the complement of the marker sequence to form a

ES 2 358 296 T3 hybrid primer oligonucleotide: first RNA product so that a primer extension reaction can be initiated from the primer oligonucleotide; extending the primer oligonucleotide in a primer extension reaction with a DNA polymerase to produce a second primer extension product complementary to the first RNA product, and the second primer extension product has a 3 'end that is complementary to a 5 'end of the first RNA product; separating the second primer extension product from the first RNA product by using an enzyme that selectively degrades said first RNA product; treating the second primer extension product with the promoter oligonucleotide to form a promoter oligonucleotide: second primer extension hybrid; extending a 3 'end of the second primer extension product in the promoter oligonucleotide: second primer extension product hybrid to add a complementary sequence to the second region of the promoter oligonucleotide; and transcribing from the promoter oligonucleotide hybrid: second primer extension product multiple copies of a second RNA product complementary to the second primer extension product using RNA polymerase, wherein the base sequence of the second primer extension product RNA is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the marker sequence.

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

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

The promoter oligonucleotide hybridized to the first primer extension product is extended with a DNA polymerase to produce a primer extension product complementary to the first primer extension product; and the promoter oligonucleotide hybridized to said second primer extension product is extended with a DNA polymerase to produce a primer extension product complementary to the second primer extension product.

The separation steps of the described methods can be carried out with a ribonuclease activity provided by DNA polymerase. Alternatively, the separation steps are carried out with a ribonuclease activity provided by an enzyme other than said DNA polymerase.

According to another embodiment of the present disclosure, the target nucleic acid sequence is an RNA target sequence, and step (c) comprises: extending the labeled oligonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction with a DNA polymerase to produce a first primer extension product comprising a region complementary to the target nucleic acid sequence, wherein the oligonucleotide labeling further comprises a third region located 5 'to the second region, and the third region comprises a promoter for an RNA polymerase; separating the first primer extension product from the target nucleic acid by using an enzyme that selectively degrades that part of the target nucleic acid hybridized to the first primer extension product; treat the first primer extension product with the first oligonucleotide, and the first oligonucleotide is a primer oligonucleotide that hybridizes to a region of the first primer extension product that is complementary to a 5 'end of the target nucleic acid sequence to forming a primer oligonucleotide: first primer extension product hybrid so that a primer extension reaction can be initiated from the primer oligonucleotide; extending the primer oligonucleotide in a primer extension reaction with a DNA polymerase to produce a second primer extension product complementary to the first primer extension product; and using the second primer extension product as a template to transcribe multiple copies of a first RNA product complementary to at least a portion of the second primer extension product by using an RNA polymerase that recognizes the promoter and initiates transcription to from him, wherein the base sequence of the first RNA product is substantially identical to the base sequence of the marker sequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, step (c) further comprises: treating the first RNA product with the primer oligonucleotide to form a primer oligonucleotide: first RNA product hybrid so that a primer extension reaction can be initiated. from the priming oligonucleotide; extending the primer oligonucleotide in a primer extension reaction with a DNA polymerase to produce a third primer extension product complementary to the first RNA product, and the third primer extension product has an end

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3 'which is complementary to a 5' end of the first RNA product; separating the third primer extension product from the first RNA product by using an enzyme that selectively degrades the first RNA product; treating the third primer extension product with the second oligonucleotide, and the second oligonucleotide is a promoter oligonucleotide comprising first and second regions, and the first region comprising a hybridization sequence that hybridizes to the complement of the marker sequence to form a hybrid oligonucleotide promoter: third primer extension product so that a primer extension reaction can be initiated from the promoter oligonucleotide, and the second region comprises a promoter for an RNA polymerase that is located 5 'to the first region; extending the promoter oligonucleotide in a primer extension reaction with DNA polymerase to produce a fourth primer extension product complementary to the third primer extension product; extending the third primer extension product to add a complementary sequence to the promoter; transcribing from the promoter oligonucleotide hybrid: third primer extension product multiple copies of a second RNA product complementary to the third primer extension product by using an RNA polymerase that recognizes the promoter and initiates transcription from it, wherein the base sequence of the second RNA product is substantially identical to the base sequence of the marker sequence and the complement of the target nucleic acid sequence.

In another aspect of this embodiment, the separation steps are carried out with a ribonuclease activity provided by DNA polymerase. Alternatively, the separation steps are carried out with a ribonuclease activity provided by an enzyme other than DNA polymerase.

According to another embodiment of the present disclosure, the target nucleic acid sequence is a DNA target sequence, and step (c) comprises: extending the labeled oligonucleotide hybridized to the target nucleic acid sequence in a primer extension reaction with a DNA polymerase to produce a first primer extension product comprising a region complementary to the target nucleic acid sequence; treat the first primer extension product with the first oligonucleotide, and the first oligonucleotide is a promoter oligonucleotide comprising first and second regions, and the first region comprises a hybridization sequence that hybridizes to a region of the first primer extension product that is complementary to a 5 'end of the target nucleic acid sequence to form a promoter oligonucleotide hybrid: first primer extension product, and the second region is a promoter for an RNA polymerase that is located 5 'to the first region; transcribing from the promoter oligonucleotide hybrid: first primer extension product multiple copies of a first RNA product complementary to at least a part of the first primer extension product using an RNA polymerase that recognizes the promoter and initiates transcription from him, wherein the base sequence of the first RNA product is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the marker sequence; treat the first RNA product with the second oligonucleotide, and the second oligonucleotide is a primer oligonucleotide that hybridizes to the complement of the marker sequence to form a primer oligonucleotide: first RNA product hybrid so that an extension reaction can be initiated of primer from the priming oligonucleotide; extending the primer oligonucleotide in a primer extension reaction with a DNA polymerase to provide a second primer extension product that comprises the complement of the first RNA product, and the second primer extension product has a 3 'end that is complementary to a 5 'end of the first RNA product; separating the second primer extension product from the first RNA product by using an enzyme that selectively degrades the first RNA product; treating the second primer extension product with the promoter oligonucleotide to form a promoter oligonucleotide: second primer extension hybrid; extending a 3 'end of the second primer extension product in the promoter oligonucleotide: second primer extension product to add a complementary sequence to the promoter; and transcribing from the promoter oligonucleotide hybrid: second primer extension product multiple copies of a second RNA product complementary to the second primer extension product using RNA polymerase, wherein the base sequence of the second primer extension product RNA is substantially identical to the base sequence of the target nucleic acid sequence and the complement of the marker sequence.

In one aspect of this embodiment, the promoter oligonucleotide is modified to prevent the initiation of DNA synthesis from it.

In another aspect, step (a) further comprises: treating the nucleic acid sample with a displacer oligonucleotide that hybridizes to the target nucleic acid downstream of the labeled oligonucleotide so that a primer extension reaction can be initiated from the oligonucleotide. displacer; and extending the displacer oligonucleotide in a primer extension reaction with a DNA polymerase to produce a third primer extension product that displaces said first primer extension product from the target nucleic acid.

ES 2 358 296 T3

In yet another embodiment, step (a) further comprises treating the nucleic acid sample with a binding molecule that binds the target nucleic acid adjacent or near a 5 'end of the target nucleic acid sequence, at the that the first primer extension product has a 3 'end that is determined by said binding molecule and that is complementary to the 5' end of the target nucleic acid sequence.

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

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

The separation steps, in one embodiment, are carried out by means of a ribonuclease activity provided by said DNA polymerase. Alternatively, the separation steps are carried out by a ribonuclease activity provided by an enzyme other than said DNA polymerase.

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, and the kit comprises: a labeled oligonucleotide comprising a first region comprising a target hybridizing sequence that hybridizes to a 3 'end of a target nucleic acid sequence under a first set of conditions, such that the first region can be extended in a dependent manner of the template in the presence of a DNA polymerase, and a second region comprising a marker sequence located 5 'to the first region, wherein the second region does not stably hybridize to a target nucleic acid containing the target nucleic acid sequence under the first set of conditions; a tag closure sequence that hybridizes to the target hybridization sequence under a second set of conditions, thereby blocking hybridization of the tagged oligonucleotide to the target nucleic acid sequence, where the tag closure sequence does not stably hybridizes to the target hybridization sequence under the first set of conditions; and a first primer oligonucleotide that hybridizes to the complement of the marker sequence under the second set of conditions such that the first primer oligonucleotide can be elongated in a template-dependent manner in the presence of a DNA polymerase.

In a more particular aspect of this embodiment, the labeled oligonucleotide further comprises a third region containing a promoter for an RNA polymerase, and the third region is located 5 'to the second region.

In another aspect, the 3'-terminal base of the target hybridization sequence hybridizes to a 5'-terminal base of the marker closure sequence when the target hybridization sequence is not hybridized to the target nucleic acid sequence in the second set of conditions.

In yet another aspect, the 5 'end of the tag closure sequence includes a moiety to stabilize a double-stranded molecule formed between the tag closure sequence and the target hybridizing sequence when the target hybridizing sequence is not hybridized to the target nucleic acid sequence in the second set of conditions.

In another aspect, the tagged oligonucleotide and the tag closure sequence constitute different molecules, and the tag closure sequence is a tag closure oligonucleotide. Alternatively, the tagged oligonucleotide and the tag closure sequence are contained in the same molecule.

The tag closure sequence can be linked to the tagged oligonucleotide by a non-nucleotide spacer, for example a non-nucleotide spacer comprising at least one of abasic nucleotides and polyethylene glycol.

In another aspect, a 3 'end of the tag closure sequence is attached to a 5' end of the tagged oligonucleotide. Alternatively, a 5 'end of the tag closure sequence is attached to a 5' end of the tagged oligonucleotide.

In yet another aspect, the tag closure sequence hybridizes to the target hybridizing sequence to form an antiparallel double stranded molecule when the target hybridizing sequence is not hybridizing to the target nucleic acid sequence under the second set of conditions.

ES 2 358 296 T3

In another aspect, the tag closure sequence is modified to prevent initiation of DNA synthesis from it, for example by including a blocking moiety located at its 3 'end.

In another aspect, the tag closure sequence hybridizes to the target hybridization sequence to form a parallel double stranded molecule when the target hybridization sequence is not hybridized to the target nucleic acid sequence under the second set of conditions.

In yet another aspect, the double-stranded molecule comprises a 3'-terminal base of the target hybridization sequence hybridized to a 3'-terminal base of the tag closure sequence.

The marker closure sequence, in this regard, can be modified to prevent initiation of DNA synthesis from it, for example by including a blocking moiety located at its 3 'end.

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

In another aspect, the kit further comprises a second primer oligonucleotide that hybridizes to the complement of a 5 'end of the target nucleic acid sequence under the second set of conditions so that the second primer oligonucleotide can be extended in a dependent manner. of the template in the presence of a DNA polymerase.

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

The promoter oligonucleotide, in this regard, can be modified to prevent initiation of DNA synthesis from it, for example by including a blocking moiety located at its 3 'end.

In yet another aspect, the promoter oligonucleotide can be elongated in a template-dependent manner in the presence of a DNA polymerase when the hybridizing sequence is hybridized to the complement of the 5 'end of the target nucleic acid sequence under the second set of conditions.

Kits may further comprise one or more reagents or components selected from one or more of a DNA polymerase (such as a reverse transcriptase), an RNA polymerase, nucleoside triphosphates, a solid support for the binding of a complex comprising the nucleic acid target and labeled oligonucleotide. In another aspect, the labeled oligonucleotide is free in solution.

In another aspect, the kit does not include a restriction enzyme capable of cleaving a double-stranded molecule formed between the tag closure sequence and the target hybridization sequence under the second set of conditions.

In yet another aspect, the target hybridization sequence hybridizes to a 3 'end of multiple target nucleic acid sequences under the first set of conditions.

In another embodiment, the labeled oligonucleotide is a universal bacterial oligonucleotide or a universal fungal oligonucleotide. For example, in one aspect, the target hybridization sequence hybridizes to the target region at a 3 'end of one or more target nucleic acid sequences, and the target region is present in a variety of microorganisms belonging to a class of microorganisms selected from the group consisting of Eubacteria, gram-positive bacteria, gram-negative bacteria, and fungi in the first set of conditions. In another embodiment, said one or more target nucleic acid sequences are ribosomal nucleic acid sequences.

In a more particular aspect, the microorganisms belong to a class of bacterial microorganisms selected from the group consisting of Staphylococci spp. (eg, Staphylococcus aureus, Staphylococcus epidermis, and Staphylococcus haemolyticus), Streptococci spp. (eg, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Viridans streptococci, and beta-hemolytic streptococci), Enterococcus spp. (eg, Enterococcus faecium and Enterococcus faecalis), Escherichia spp. (eg, Escherichia coli), Klebsiella spp. (eg, Klebsiella pneumoniae and Klebsiella oxytoca), Pseudomonas spp. (eg, Pseudomonas aeruginosa), Enterobacter spp. (eg, Enterobacter cloacae and Enterobacter aerogenes), Proteus spp. (eg, Proteus mirabilis), Bacterioides spp., Clostridium spp., Serratia spp. (eg, Serratia marcescens), Acinetobacter spp. (eg, Acinetobacter baumannii) and Stenotrophomonas spp. (eg, Stenotrophomonas maltophilia). At least some of these microorganisms would be suitable for detection in a sepsis test.

ES 2 358 296 T3

In another particular aspect, the microorganisms belong to a fungal group of microorganisms selected from the group consisting of Candida spp. (eg, Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida lusitaniae, Candida krusei, Candida zeylanoides, Candida guilliermondi, Candida pseudotropicalis, and Candida famata), Histoplama capsulatum, Cryptococcus spp. (eg, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii) Coccidioides spp. (eg, Coccidioides immitis), Trichosporon spp. (eg, Trichosporon cutaneum), Malassezia spp. (eg, Malassezia furfur), Rhodotorula spp., Nocardia spp. (eg, Nocardia asteroides), Fusarium spp. and Asperigillus spp. (eg, Asperigillus fumigatus). At least a portion of these microorganisms would be suitable for detection in a sepsis test.

According to another embodiment of the disclosure, a reaction mixture is provided for amplifying a target nucleic acid sequence, and the reaction mixture comprises: a labeled oligonucleotide comprising first and second regions, and the first region comprises a target hybridization sequence hybridized to a 3 'end of a target nucleic acid sequence and the second region comprises a marker sequence located 5' to the sequence hybridization to the target; a first oligonucleotide comprising a hybridizing sequence that hybridizes to a 3 'end of the complement of the target nucleic acid sequence; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of the marker sequence, wherein the unhybridized labeled oligonucleotide in the reaction mixture has an inactive form that blocks or prevents the unhybridized labeled oligonucleotide from hybridizing to the sequence. of target nucleic acid.

In a more particular aspect according to this embodiment, the inactive form of the labeled oligonucleotide comprises a marker closure sequence hybridized to the target hybridization sequence.

In another aspect, the tagged oligonucleotide and said tag closure sequence are different molecules, and the tag closure sequence is a tag closure oligonucleotide.

In another aspect, the tagged oligonucleotide and the tag closure sequence are contained in the same molecule.

In yet another aspect, the labeled oligonucleotide is not attached to a solid support.

Other embodiments of the description relate to the use of the methods described herein as a means of monitoring bioprocess samples, 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) treating a first bioprocess sample with a labeled oligonucleotide, wherein said labeled oligonucleotide comprises first and second regions, and the first region comprises a target hybridization sequence capable of hybridizing to a target nucleic acid sequence of an organism of interest, and the second region comprises a marker sequence, located 5 'to said target hybridizing sequence, that does not stably hybridize to the target nucleic acid sequence; under conditions where the labeled oligonucleotide stably hybridizes to the target nucleic acid sequence present in said first sample; (b) removing or inactivating the unhybridized labeled oligonucleotide from the first bioprocess sample; and (c) exposing a second bioprocess sample, and the second bioprocess sample comprises the first bioprocess sample, and further comprises additional bioprocess samples, to reagents and amplification conditions sufficient for amplification of the target nucleic acid sequence by the use of: (i) a first oligonucleotide that hybridizes to a complement of the marker sequence and (ii) a second oligonucleotide sequence that hybridizes to a complement of the target nucleic acid sequence, wherein the detectable amplification resulting from the first and second oligonucleotides is contributed by the target nucleic acid sequence of an organism of interest in the first bioprocess sample, and not by the target nucleic acid sequence contributed by the additional bioprocess samples.

In another embodiment, the present disclosure provides a method of monitoring a bioprocess for the presence of a contaminating nucleic acid comprising the steps of (a) treating a first bioprocess sample with a first labeled oligonucleotide, wherein the first oligonucleotide marking comprises first and second regions, and the first region comprises a target hybridization sequence capable of hybridizing to a target nucleic acid sequence of an organism of interest and the second region comprises a first marker sequence, located 5 'to the target hybridization sequence, which does not stably hybridize to the target nucleic acid sequence; under conditions where the first labeled oligonucleotide stably hybridizes to the target nucleic acid sequence present in said first sample; (b) treating a second bioprocess sample with a second labeled oligonucleotide, wherein the second labeled oligonucleotide comprises first and second regions, and the first region comprises a target hybridization sequence capable of hybridizing to the target nucleic acid sequence of the organism of interest and the second region comprises a second marker sequence, located 5 'to the target hybridization sequence and different from the first marker sequence, which does not

ES 2 358 296 T3 stably hybridizes to target nucleic acid sequence; under conditions where the second labeled oligonucleotide stably hybridizes to the target nucleic acid sequence present in the second sample; and (c) carrying out a nucleic acid amplification reaction in a third bioprocess sample, and the third bioprocess sample comprises the first and second bioprocess sample, by using: (i) a first oligonucleotide that hybridizes to a complement of the first marker sequence; (ii) a second oligonucleotide sequence that hybridizes to a complement of the second marker sequence; and (iii) a third oligonucleotide that hybridizes to a complement of the target nucleic acid sequence; wherein the detection of the amplification product resulting from the first and second oligonucleotides is indicative of the presence of the target nucleic acid sequence of the organism of interest in the first bioprocess sample, and wherein the detection of the amplification product that resulting from the first and third oligonucleotides is indicative of the presence of the target nucleic acid sequence of the organism of interest in the second bioprocess sample.

In a further embodiment of the disclosure, a preamplification reaction mixture is provided for the selective amplification of one or more target nucleic acid sequences, wherein the reaction mixture comprises: a labeled oligonucleotide comprising first and second regions, and said first region comprises a target hybridization sequence hybridized to a target region contained at a 3 'end of one or more target nucleic acid sequences present in the reaction mixture and the second region comprises a marker sequence located 5 'to the target hybridization sequence; a first oligonucleotide comprising a hybridizing sequence that hybridizes to a 3 'end of the complement of one or more of the target nucleic acid sequences; and a second oligonucleotide comprising a hybridization sequence that hybridizes to the complement of the marker sequence, wherein the second oligonucleotide preferably does not stably hybridize to a target nucleic acid containing the target nucleic acid so that it can be enzymatically extended in the presence of a nucleic acid polymerase added to the reaction mixture to produce an extension product primer complementary to one or more of the target nucleic acid sequences, wherein the reaction mixture is substantially free of an active form of the labeled oligonucleotide that is not hybridized to the target region contained in one or more target nucleic acid sequences present in the reaction mixture, wherein the active form of the oligonucleotide labeled has a target hybridization sequence available for hybridization to the target region present in a non-target nucleic acid added to the reaction mixture, and wherein the reaction mixture does not include a nucleic acid polymerase capable of elongating any of the oligonucleotides in a template-dependent manner. The non-target nucleic acid is from a source external to 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 can be environmental, or it can be a component or reagent added to the reaction mixture, such as a nucleic acid polymerase. The tagged oligonucleotide can be a tagged primer oligonucleotide or a tagged promoter oligonucleotide having a promoter recognized by an RNA polymerase located 5 'to the second region.

In one aspect of this embodiment, the labeled primer oligonucleotide does not include a promoter for RNA polymerase, while the first oligonucleotide includes an RNA promoter located 5 'to the hybridization sequence of the first oligonucleotide. In a preferred aspect, the first oligonucleotide further includes a blocking moiety located at its 3 'end. In another aspect that is useful for amplifying a target sequence of E. coli, the target hybridization sequence of the labeled oligonucleotide consists of SEQ ID NO: 19, its RNA equivalent or a complement thereof, and the first hybridization sequence of the first oligonucleotide consists of SEQ ID NO: 20, its equivalent of RNA or a complement thereof.

In another aspect of this embodiment, the tagged oligonucleotide and the first oligonucleotide may each include a promoter for an RNA polymerase located 5 'to the tag sequence and the hybridization sequence, respectively. In this aspect, the first oligonucleotide can include a blocking moiety located at its 3 'end.

In yet another aspect of this embodiment, the tagged oligonucleotide includes a tag closure sequence attached to its 3 'end, whereby a unitary molecule called a tag molecule is formed. Depending on the nature of the amplification reaction, the marker molecule may or may not include a promoter for an RNA polymerase located 5 'to the marker sequence. In one embodiment, marker molecules that have not hybridized to the target region of at least one target nucleic acid sequence remain free in the reaction mixture (i.e., the marker molecules do not form hybrid double-stranded molecules by means other than self- hybridization). Self-hybridizing marker molecules are called hairpin marker molecules, which are an inactive form of the marker molecule that prevents it from hybridizing to any complementary nucleic acid that is subsequently added to the reaction mixture, such as by means of a contaminated enzyme preparation. or reagent containing non-target nucleic acids. In yet another aspect of this embodiment, substantially all of the marker molecules in the reaction mixture are in a hybridized state (hybridized to the target region of a sequence of

ES 2 358 296 T3 target nucleic acid or themselves as hairpin marker molecules). At least a portion of the marker molecules that have not hybridized to the target region of a target nucleic acid sequence (i.e. hairpin marker molecules) are removed from the reaction mixture, for example by subjecting the reaction mixture to a target capture and wash procedure.

In yet a further aspect of this embodiment, there are substantially no labeled oligonucleotides that exist in an unhybridized state when the reaction mixture is exposed to an enzyme preparation to amplify one or more target nucleic acid sequences. Thus, in this regard, the unhybridized labeled oligonucleotides specific for one or more target nucleic acid sequences provided by the sample of interest have been substantially removed from the reaction mixture. This can be accomplished, for example, with a target capture and wash procedure that separates the hybridized labeled oligonucleotides from the unhybridized labeled oligonucleotides, and then selectively removes the unhybridized labeled oligonucleotides from the reaction mixture.

In yet another aspect of this embodiment, the labeled oligonucleotide does not include a label closure sequence or a label closure oligonucleotide. Therefore, in this regard the labeled oligonucleotide cannot be characterized as a marker molecule.

In yet another aspect of this embodiment, a probe is included to detect a synthesized amplification product in an in vitro reaction involving enzymatic extension of the labeled oligonucleotide and the second oligonucleotide. The amplification product includes copies of one or more of the target nucleic acid sequences and / or their complements.

In yet another embodiment, a reaction mixture is provided for the selective amplification of one or more target nucleic acid sequences, wherein the reaction mixture comprises: a labeled oligonucleotide comprising first and second regions, wherein the first region comprises a target hybridization sequence hybridized to a 3 'end of a target nucleic acid sequence, and the second region comprises a marker sequence located 5' to of the hybridization sequence to the target; a first oligonucleotide comprising a hybridizing sequence that hybridizes to a 3 'end of the complement of the target nucleic acid sequence; and a second oligonucleotide comprising a hybridizing sequence that hybridizes to the complement of the marker sequence, wherein the second oligonucleotide preferably does not stably hybridize to a target nucleic acid containing the target nucleic acid, such that it can be enzymatically extended in the presence of a nucleic acid polymerase added or present in the reaction mixture to produce a primer extension product complementary to one or more of the target nucleic acid sequences, and wherein substantially all of the unhybridized tagged oligonucleotide in the reaction mixture has an inactive form that blocks or prevents said unhybridized tagged oligonucleotide from hybridizing to the target nucleic acid sequence.

The inactive form of the labeled oligonucleotide may comprise a marker closure sequence hybridized to the target hybridization sequence. The tag closure sequence may be a different molecule when not hybridized to the target hybridization sequence, or it may be contained in a molecule that includes the tagged oligonucleotide, in which case the tag closure sequence is preferably end-attached. 5 'of the oligonucleotide labeled by a non-nucleotide spacer (ie, the spacer components cannot be copied by a nucleic acid polymerase). The labeled oligonucleotide may or may not be attached to a solid support, and is preferably not directly attached to the solid support (eg, particles or spheres). If attached to a solid support, directly or indirectly, the labeled oligonucleotide can further function as a capture probe for the binding and immobilization of a target nucleic acid sequence.

The labeled oligonucleotides of the above reaction mixture embodiments can possess the characteristics of any of the various labeled oligonucleotide embodiments described below. And, unless specifically excluded, reaction mixtures may further include the reagents and components necessary to carry out an amplification reaction.

These and other features and advantages of the present disclosure will become apparent upon reference to the following detailed description, the accompanying drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the steps of a transcription-based amplification reaction initiated with a labeled primer oligonucleotide that hybridizes to a 3 'end of a target RNA sequence. A first extension product formed with the labeled primer oligonucleotide has a 3 'end that is determined by a hybridized terminator oligonucleotide adjacent to or close to the 5' end of the RNA target sequence. A blocked promoter oligonucleotide hybridizes to a 3 'end of the first extension product and is used to generate RNA transcripts that cyclically participate in the amplification reaction.

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

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

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

FIG. 5 illustrates the steps of a transcription-based amplification reaction initiated with a labeled primer oligonucleotide that hybridizes to a 3 'end of a single-stranded DNA target sequence. A first extension product formed with the labeled primer oligonucleotide has a 3 'end that is determined by a hybridized terminator oligonucleotide adjacent to or close to the 5' end of the target DNA sequence. A displacer oligonucleotide hybridized 5 'to the labeled primer oligonucleotide is extended to form a second extension product that displaces the first extension product from the target DNA sequence. A blocked promoter oligonucleotide hybridizes to a 3 'end of the first extension product and is used to generate RNA transcripts that cyclically participate in the amplification reaction.

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

FIG. 7 illustrates the steps of the polymerase chain reaction that is initiated with a labeled primer oligonucleotide that hybridizes to a target DNA sequence.

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

FIG. 9 illustrates the steps of a reverse transcription polymerase chain reaction initiated with a labeled primer oligonucleotide that hybridizes to a target RNA sequence.

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

FIG. 11 illustrates a discrete 3 'blocked tag closure oligonucleotide hybridized in an antiparallel manner to the 3' end of a tagged primer oligonucleotide, thereby blocking hybridization of the tagged primer oligonucleotide to a target nucleic acid sequence.

FIG. 12 illustrates a discrete 3 'blocked marker closure oligonucleotide hybridized in an antiparallel manner to the 3' end of a tagged promoter oligonucleotide, thereby blocking hybridization of the tagged promoter oligonucleotide to a target nucleic acid sequence.

FIG. 13 illustrates a hairpin tag molecule that includes a 3 'blocked tag closing sequence hybridized in a parallel fashion to the 3' end of a tagged primer oligonucleotide, thereby blocking the hybridization of the tagged primer oligonucleotide to a sequence of target nucleic acid. A 5 'end of the tag closure sequence is linked to the 3' end of a primer sequence tagged primer oligonucleotide by a non-nucleotide spacer.

FIG. 14 illustrates a hairpin tag molecule that includes a 3 'blocked tag closing sequence hybridized in a parallel fashion to the 3' end of a tagged promoter oligonucleotide, thereby blocking hybridization of the tagged promoter oligonucleotide to an acid sequence target nucleic. A 5 'end of the tag closure sequence is linked to the 3' end of a promoter sequence of the promoter oligonucleotide tagged by a non-nucleotide spacer.

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 primer oligonucleotide, thereby blocking hybridization of the tagged primer oligonucleotide to a sequence of target nucleic acid. A 5 'end of the tag closure sequence is linked to the 3' end of a primer sequence tagged primer oligonucleotide by a non-nucleotide spacer.

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 to an acid sequence. target nucleic. A 5 'end of the tag closure sequence is linked to the 3' end of a promoter sequence of the promoter oligonucleotide tagged by a non-nucleotide spacer.

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FIG. 17 shows the crude curves for the HCV applications where no target was added in the amplification reagent. There was no detectable amplification when the HCV transcript was not added to the target capture or amplification reagents, while the mean time for reactions containing 1 x 10⁶ copies of the HCV transcript in the target capture reagent was 6.3 minutes.

FIG. 18 shows the crude curves for HCV amplifications in which target was added in the amplification reagent. There was no detectable amplification when the HCV transcript was added in the amplification reagent, while the mean time for reactions containing 1 x 10⁶ copies of the HCV transcript in the target capture reagent was 6.3 minutes. Zero samples in the target capture did not amplify, even with 1 million copies of HCV 1a added in the amplification reagent.

FIG. 19 shows the crude curves for the HCV amplifications in which target and non-T7 labeled primer were added in the amplification reagent. The mean time for 1 million copies of HCV 1a target present only in the step of capturing the target with non-T7 labeled primer and terminator oligonucleotide added in the amplification reagent was 7.2 minutes. Zero samples in capturing the target with target, terminator oligonucleotide, and non-T7 labeled primer in the amplification reagent also produced solid amplification, with a mean time = 8.6 minutes.

FIG. 20 is a graph showing the results of time-dependent monitoring of nucleic acid amplification reactions that included 0 or 10⁶ copies of a synthetic E. coli rRNA template. The thin broken line shows the results for the reaction carried out by using 0 template copies, and the thick solid line shows the results for the reaction carried out by using 10⁶ mold copies.

FIG. 21 is a graph showing the results of time-dependent monitoring of nucleic acid amplification reactions that included 0.10³ or 10⁵ copies of a synthetic E. coli rRNA template.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, nucleic acid amplification methods are provided that desirably reduce or eliminate false positive amplification signals that result from contaminating biological material that may be present in a reagent or component of an amplification reaction. The methods provided also allow for less stringent purification and / or sterility efforts than have been conventionally required to ensure that the enzymes and other reagents or components used in the amplification reactions, and the medium in which they are carried out carried out amplification reactions, are free from contamination by microorganisms or their components, such as nucleic acid material, that can provide false positive results.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, recombinant DNA, and chemistry, which are within the skill of the art. Such techniques are fully explained in the literature. See, eg, Molecular Cloning A Laboratory Manual, 2^to Ed., Sambrook et al., Ed., 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., Pat. US No. 4,683,195; Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984); B. Perbal, A Practical Guide To Molecular Cloning (1984); 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

The following expressions have the following meanings, unless expressly stated otherwise. It should be noted that the term an entity refers to one or more of that entity; for example, a nucleic acid is understood to represent one or more nucleic acids. As such, the terms one (or one), one or more, and at least one can be used interchangeably herein.

Nucleic acid

The term "nucleic acid" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to any chain of two or more nucleotides, nucleosides, or nucleobases (eg, deoxyribonucleotides or ribonucleotides) covalently linked together. . Nucleic acids include, but are not limited to, viral genomes, or portions thereof, DNA or RNA, bacterial genomes, or portions thereof, fungal, plant, or animal genomes, or portions thereof, messenger RNA. (mRNA), ribosomal RNA (rRNA), transfer RNA

ES 2 358 296 T3 (tRNA), plasmid DNA, mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid can be provided in a linear (eg, mRNA), circular (eg, plasmid), or branched form, as well as in a double-stranded or single-stranded form. Nucleic acids can include modified bases to alter nucleic acid function or behavior, eg, the addition of a 3'-terminal dideoxynucleotide to block the addition of additional nucleotides to the nucleic acid. As used herein, a nucleic acid sequence refers to the sequence of bases that constitutes a nucleic acid. The term polynucleotide can be used herein to denote a nucleic acid strand. Throughout this application, nucleic acids are indicated to have a 5 'end and a 3' end. Standard nucleic acids, eg, DNA and RNA, are generally synthesized 3 'to 5', that is, by adding nucleotides to the 5 'end of a growing nucleic acid.

A nucleotide is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon carbohydrate, and a nitrogen base. The 5-carbon carbohydrate found in RNA is ribose. In DNA, the 5-carbon carbohydrate is 2'-deoxyribose. The term also includes analogs of such subunits, such as a 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 residue, and a uracil at the base position of the nucleotide.

A non-nucleotide unit is a unit that does not participate significantly in the hybridization of a polymer. Such units, for example, should not participate in significant hydrogen bonding with a nucleotide, and would exclude units that have one of the five nucleotide bases or analogs thereof as a component.

Target Nucleic Acid / Target Sequence

A target nucleic acid is a nucleic acid present in a nucleic acid sample that comprises a target sequence to be amplified. Target nucleic acids can be DNA or RNA, as described herein, and can be single-stranded or double-stranded. The target nucleic acid can include other sequences in addition to the target sequence that may not be amplified. Typical target nucleic acids include viral genomes, bacterial genomes, fungal genomes, plant genomes, animal genomes, rRNAs, tRNAs, or mRNAs from viruses, bacteria, or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.

Target nucleic acids can be isolated from any number of sources based on the purpose of the amplification assay to be performed. Sources of the target nucleic acids include, but are not limited to, clinical samples (e.g., blood, either whole blood or platelets, urine, saliva, feces, semen, or cerebrospinal fluid), environmental samples (e.g. , water or soil samples), food samples, beverages, industrial samples (eg, products and process materials, including water), seed stocks, cDNA libraries, or total cellular RNA. Isolated means that a sample containing a target nucleic acid is taken from its natural environment; however, the term does not connote any particular degree of purification. If necessary, the target nucleic acids of the present invention are made available for interaction with the various oligonucleotides of the present invention. This may include, for example, lysis of cells or permeabilization of cells to release the target nucleic acid from the cells, which may then be followed by one or more purification steps, such as a series of isolation and washing steps. . See, eg, Clark et al., Method for Extracting Nucleic Acids from a Wide Range of Organisms, pat. U.S. No. 5,786,208; and Hogan, Polynucleotide Matrix-Based Method of Identifying Microorganisms, pat. U.S. No. 6,821,770. This can be especially important when the source of the sample or cellular material released into the sample can 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. The target nucleic acids of the present invention can be purified to some extent prior to the amplification reactions described herein, but in other cases the sample is added to the amplification reaction without any additional manipulation.

The term "target sequence" refers to the particular nucleotide sequence of the target nucleic acid to be amplified. The target sequence includes the complexing sequences to which oligonucleotides (eg, labeled oligonucleotides, primer oligonucleotides, and / or promoter oligonucleotides) complex during the methods of the present invention. When the target nucleic acid is originally single stranded, the term "target sequence" will also refer to the 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 the sense (+) and antisense (-) strands. In choosing a target sequence, the skilled artisan will understand that a unique sequence should be chosen to distinguish between unrelated or closely related target nucleic acids. As those of ordinary skill in the art will understand, unique sequences are judged from the analysis medium. At least the sequences recognized by the target hybridization sequence of a labeled oligonucleotide and the associated detection probe (s) (as described in more detail elsewhere

ES 2 358 296 T3 part herein) should be unique in the medium being analyzed, but need not be unique within the universe of all possible sequences. Furthermore, although the target sequence should contain a unique sequence for recognition by a labeled oligonucleotide or detection probe, it is not always the case that the primer oligonucleotide and / or the promoter oligonucleotide recognize unique sequences. In certain embodiments, it may be desirable to choose a target sequence that is common to a class of organisms, for example, a sequence that is common to all E. coli strains that might be in a sample. In other situations, a highly specific target sequence, or a target sequence having at least one highly specific region recognized by the detection probe, would be chosen to distinguish between closely related organisms, for example, E. pathogenic and non-pathogenic coli. A target sequence of the present invention can be of any practical length. A minimal target sequence includes a region that hybridizes to the target hybridization sequence of a labeled oligonucleotide, the complement of a region that hybridizes to a primer oligonucleotide or the hybridization region of a promoter oligonucleotide, and a region used for detection , eg, a region (or the complement thereof) that hybridizes to a detection probe, as described in more detail elsewhere herein. The region that hybridizes to the detection probe may overlap or be contained in the region that hybridizes to the primer oligonucleotide (or its complement) or the hybridization region to the promoter oligonucleotide (or its complement). In addition to the minimum requirements, the optimal length of a target sequence depends on several considerations, for example, the amount of secondary structure, or the self-hybridizing regions in the sequence. Determination of the optimal length is easily carried out by those of ordinary skill in the art using routine optimization methods. In general, the target sequences of the present invention range in length from about 100 nucleotides to about 150 to about 250 nucleotides. The optimal or preferred length may vary under different conditions, which can be readily analyzed by one of ordinary skill in the art according to the methods described herein. The term "amplicon" refers to a nucleic acid molecule generated during an amplification procedure that is substantially complementary or identical to a sequence contained within the target sequence. The term "amplification product" refers to an amplicon or some other product indicative of an amplification reaction.

Oligonucleotides

As used herein, the term "oligonucleotide or oligo or oligomer" is intended to encompass an oligonucleotide in the singular, as well as oligonucleotides in the plural, and refers to any polymer of two or more nucleotides, nucleosides, nucleobases, or related compounds used as a reagent. in the amplification methods of the present invention, as well as in subsequent detection methods. The oligonucleotide can be DNA and / or RNA and / or analogs thereof. The term oligonucleotide does not indicate any particular function for the reagent, and is instead used generically to cover all of those reagents described herein. An oligonucleotide can fulfill a number of different functions, e.g. it can function as a primer if it is capable of hybridizing to a complementary strand and can furthermore be prolonged in the presence of a nucleic acid polymerase, it can provide a promoter if it contains a recognized sequence by an RNA polymerase and enables transcription, and may function to prevent hybridization or prevent primer extension if appropriately positioned and / or modified. Specific oligonucleotides of the present invention are described in more detail below. As used herein, an oligonucleotide can be virtually any length, and is limited only by its specific function in the amplification reaction or in the detection of an amplification product from the amplification reaction.

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

Oligonucleotides can be modified in any way, as long as a given modification is compatible with the desired function of a given oligonucleotide. One of ordinary skill in the art can easily determine whether a given modification is suitable or desired for any given oligonucleotide of the present invention. Modifications include base modifications, carbohydrate modifications, or backbone modifications. Base modifications include, but are not limited to, the use of the following bases in addition to adenine, cytidine, guanosine, thymine, and uracil: C-5 propine, 2-amino adenine, 5-methyl cytidine, inosine, and the bases dP and dK. The carbohydrate groups of the nucleoside subunits can be ribose, deoxyribose, and the analogs thereof, including, for example, ribonucleosides having a 2'-Omethyl (2'-O-ME) substitution on the ribofuranosyl residue. . See Becker et al., Method for Amplifying Target Nucleic Acids Using Modified Primers, pat. U.S. No. 6,130,038. Other carbohydrate modifications include, but are not limited to, the 2'-amino, 2'-fluoro, (L) -alpha-threofuranosyl, and

ES 2 358 296 T3 pentopuranosyl. Nucleoside subunits can be linked by linkages such as phosphodiester linkages, modified linkages, or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleic acid sequence. Modified linkages include those linkages in which a standard phosphodiester linkage is substituted for a different linkage, such as a phosphorothioate linkage or a methylphosphonate linkage. The nucleobase subunits can be linked, for example, by replacing the natural deoxyribose phosphate backbone of DNA with a pseudo-peptide backbone, such as a 2-aminoethylglycine backbone that couples the nucleobase subunits via a carboxymethyl spacer to the central secondary amine (DNA analogs having a pseudo-peptide backbone are commonly referred to as peptide nucleic acids or PNAs, and are described in Nielsen et al., Peptide Nucleic Acids, pat. U.S. No. 5,539,082). Other linkage modifications include, but are not limited to, morpholino linkages.

Non-limiting examples of oligonucleotides or oligomers contemplated by the present invention include nucleic acid analogs containing bicyclic and tricyclic nucleoside and nucleotide analogs (LNAs). See Imanishi et al., Bicyclonucleoside and Oligonucleotide Analogues, pat. US No. 6,268,490; and Wengel et al., Oligonucleotide Analogues, pat. U.S. No. 6,670,461). The present invention contemplates any nucleic acid analog as long as the modified oligonucleotide can perform its desired function, eg, hybridize to a target nucleic acid under hybridization conditions or under stringent amplification conditions, or interact with a target nucleic acid. DNA or RNA polymerase, whereby prolongation or transcription begins. In the case of detection probes, the modified oligonucleotides must also be capable of preferentially hybridizing to the target nucleic acid under stringent hybridization conditions.

Although the design and sequence of the oligonucleotides for the present invention depend on their function as described below, a number of variables must generally be taken into account. Among the most critical are: length, melting temperature (Tm), specificity, complementarity with other oligonucleotides in the system, G / C content, polypyrimidine (T, C) or polypurine (A, G), and the sequence of the 3 'end. Controlling these and other variables is a common and well-known aspect of oligonucleotide design, and various computer programs are readily available to screen large numbers of potential oligonucleotides for the optimal ones.

The 3 'end of an oligonucleotide (or other nucleic acid) can be blocked in a variety of ways through the use of a blocking moiety, as described below. A blocked oligonucleotide is not efficiently extended by the addition of nucleotides to its 3 'end, by a DNA or RNA-dependent DNA polymerase, to produce a complementary strand of DNA. As such, a blocked oligonucleotide cannot be a primer.

As used in this description, the phrase 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 oligonucleotide, as a basic and new feature, is capable of stably hybridizing to a nucleic acid having the exact complement of one of the group's listed nucleic acid sequences under stringent hybridization conditions. An exact complement includes the corresponding DNA or RNA sequence.

The phrase an oligonucleotide that substantially corresponds to a nucleic acid sequence means that the referenced oligonucleotide is sufficiently similar to the reference nucleic acid sequence that the oligonucleotide has similar hybridization properties to the nucleic acid sequence. reference as it would hybridize to the same target nucleic acid sequence under stringent hybridization conditions.

One skilled in the art will understand that the substantially corresponding oligonucleotides of the invention may vary from the referenced sequence and still hybridize to the same target nucleic acid sequence. This nucleic acid variation can be indicated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe or primer and its target sequence. Thus, an oligonucleotide of the present invention corresponds substantially to a reference nucleic acid sequence if these percentages of base identity or complementarity are 100% to about 80%. In preferred embodiments, the percentage is from 100% to about 85%. In the most preferred embodiments, this percentage can be from 100% to about 90%; in other preferred embodiments, this percentage is from 100% to about 95%. One of skill in the art will understand the various modifications of hybridization conditions that might be necessary at the various percentages of complementarity to allow hybridization to a specific target sequence without causing an unacceptable level of nonspecific hybridization.

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Labeled Oligonucleotide / Heterologous Marker Sequence

A labeled oligonucleotide, as used herein, refers to an oligonucleotide comprising at least a first region and a second region, wherein the first region comprises a target hybridizing sequence that hybridizes to the 3 'end of a target nucleic acid sequence of interest, and wherein the second region comprises a marker sequence located 5 'to the target hybridizing sequence and which does not hybridize to or stably binds to a target nucleic acid containing the target nucleic acid sequence. Hybridization of the target hybridizing sequence to the target nucleic acid sequence produces a labeled target nucleic acid sequence. The characteristics and design considerations for the target hybridization sequence component would be the same as for the priming oligonucleotides discussed below.

The marker sequence or heterologous marker sequence can be essentially any heterologous sequence as long as it does not stably hybridize to the target nucleic acid sequence of interest and thus participate in detectable amplification. The marker sequence preferably does not stably hybridize to any sequence derived from the genome of an organism being analyzed or, more particularly, to any target nucleic acid under the reaction conditions. A marker sequence that is present in a tagged oligonucleotide is preferably designed so that it does not impair or substantially interfere with the ability of the target hybridization sequence to hybridize to its target sequence. Furthermore, the marker sequence will be of sufficient length and composition so that once a complement of the marker sequence has been incorporated into an initial DNA primer extension product, a marker specific primer oligonucleotide can be used to participate in subsequent rounds of amplification as described herein. A marker sequence of the present invention is generally at least 10 nucleotides in length, and can be extended to a length of 15, 20, 25, 30, 35, 40, 50 or more nucleotides. Those skilled in the art will recognize that the design of marker sequences and labeled oligonucleotides for use in the present invention can follow any of a variety of suitable strategies, while achieving the objectives and advantages described herein.

In certain embodiments, the tagged oligonucleotide is a tagged primer oligonucleotide comprising a tag sequence and a target hybridizing sequence. In other embodiments, the tagged oligonucleotide is a tagged promoter oligonucleotide that comprises a tag sequence, a target hybridization sequence, and a promoter sequence located 5 'to the tag sequence, and is effective in initiating transcription from it.

Inactivation

The term "inactivation" means that a heterologous marker sequence is altered such that it does not stably bind to a target nucleic acid sequence under amplification conditions. In the case of an unhybridized tagged oligonucleotide, the term "inactivation" means that the tagged oligonucleotide is altered from an active conformation that allows the target hybridizing sequence to hybridize to the target nucleic acid sequence to an inactive conformation that blocks or prevents. otherwise the target hybridization sequence hybridizes to the target nucleic acid sequence. For example, an inactive conformation can be formed under stringent conditions that allow the marker closure sequence to form a stable hybrid with the target hybridizing sequence (eg, under conditions less stringent than conditions for forming a conformation). active of the labeled oligonucleotide). Unless further altered, the marker closure sequence: target hybridization sequence hybrid remains closed under amplification conditions. Alternatively, a double-stranded molecule formed between the marker closure sequence and the target hybridization sequence can be altered by an enzyme, such as a DNase, an S1 nuclease, an endonuclease, such as a restriction enzyme that cleaves a double-stranded restriction site formed between the marker closure sequence and the target hybridization sequence, a ribonuclease activity (eg, RNase H activity) to digest the RNA component (eg, target hybridization sequence) of a DNA: RNA hybrid, or an exonuclease having 3'-a-5 'or 5'-a- activity 3 'to remove nucleotides from the target hybridizing sequence hybridized to the tag closure sequence. However, to avoid exposing a sample to a potentially contaminating source of the target nucleic acid sequence, the use of enzymes to inactivate labeled oligonucleotides that have not hybridized to the target nucleic acid sequence is generally not preferred. Other means of inactivation include chemicals to alter the target hybridization sequence so that it is unable to hybridize to a target nucleic acid sequence under the amplification conditions.

Residues can be included in the target hybridization sequence to further stabilize hybrids formed between the target closure sequence and the target hybridization sequence of the labeled oligonucleotides, especially when it is anticipated that at least some of the inactive labeled oligonucleotides will be introduced into the amplification reaction mixture. The right remains

ES 2 358 296 T3 include modified nucleotides, including LNAs, 2'-O-ME ribonucleotides, 2,6-diamino purine, 5-methyl cytosine, and C-5 propynyl cytosine or uracil. Those skilled in the art will easily be able to select the number and positions of such modified nucleotides to limit local partial denaturation at the 5 'and 3' ends of the tag closure sequence and to achieve a desired hybrid melting temperature without compromising. excessive experimentation. Other suitable moieties include minor groove binding agents and pendant groups, such as purine, DABCYL, pyrine and 5'-trimethoxy stilbene CAP.

Elimination

As used herein, the term "deletion" refers to the physical separation of labeled target nucleic acid sequences from unhybridized labeled oligonucleotides. Labeled target nucleic acid sequences can be physically separated from unhybridized labeled oligonucleotides (or heterologous marker sequences) present in a nucleic acid sample by a variety of methods known to those of skill in the art. By way of example, the labeled target nucleic acid sequences can be attached to a solid support and immobilized in a nucleic acid sample while unbound material is removed. To remove unbound material, the solid support can be subjected to one or more wash / rinse steps. The washing steps are intended to remove any remaining unhybridized labeled oligonucleotides and potentially interfere with cellular material or sample material. A rinse step is generally included when the wash solution contains a component that is inhibitory to amplification when present in a sufficiently high concentration, such as a detergent. The solid support preferably binds specifically to the target nucleic acids or the labeled target nucleic acid sequences to prevent the unhybridized labeled oligonucleotide (or unbound heterologous marker sequences) from participating in the amplification reaction. Exemplary means of capturing, immobilizing, and purifying target nucleic acids are discussed below, an example of which is described in Weisburg et al., Two-Step Hybridization and Capture of a Polynucleotide, pat. U.S. No. 6,534,273.

Marker Closure Sequence / Marker Closure Oligonucleotide

The phrases tag closure sequence and tag closure oligonucleotide refer to an oligonucleotide that is complementary to a portion of the target hybridization sequence of a tagged oligonucleotide. The length and sequence of the marker closure sequence are selected such that the marker closure sequence does not stably hybridize to the target hybridization sequence of the labeled oligonucleotide under a first set of conditions that allow stable hybridization of the target hybridization sequence to a target sequence. The marker closure sequence can include abasic nucleotides or base mismatches with the target hybridizing sequence. As long as the tagged oligonucleotide is not hybridized to the target sequence, the tag closure sequence stably hybridizes to the target hybridization sequence under a second set of less stringent conditions, thereby inactivating or blocking the oligonucleotide labeled hybridizes to the target sequence. The tag closure sequence may be in the form of a discrete oligonucleotide or it may be attached to the 5 'end of a tagged oligonucleotide (tag molecule), such that it forms a hairpin structure with the tagged oligonucleotide under the second set of conditions ( hairpin marker molecule). If part of a marker molecule, the tag closure sequence is preferably linked to the tagged oligonucleotide via a non-nucleotide spacer region (eg, abasic nucleotides or polyethylene glycol) of sufficient length so that the closure sequence of the marker hybridizes to the target hybridization sequence under the second set of conditions. The marker closing sequence can be modified to prevent initiation of DNA synthesis from it, which can include a blocking moiety located at its 3 'end. The marker closure sequence is at least 3, but not more than 20 bases in length. Typical marker closure sequences are 10 to 16 bases in length.

Amplification or Amplification of Nucleic Acid

Nucleic acid amplification or amplification means the production of multiple copies of a target nucleic acid that contains at least a portion of the desired specific target nucleic acid sequence. The multiple copies can be called amplicons or amplification products. In certain embodiments, the amplified target contains less than the entire target gene sequence (introns and exons) or an expressed target gene sequence (exon splice transcript and non-translated flanking sequences). For example, specific amplicons can be produced by amplifying a portion of the target polynucleotide using amplification primers that hybridize to, and initiate polymerization from, internal positions of the target polynucleotide. Preferably, the amplified portion contains a detectable target sequence that can be detected using a variety of well-known methods.

ES 2 358 296 T3

Many well-known nucleic acid amplification methods require thermocycling to denature double-stranded nucleic acids and alternatively hybridize primers; however, other well known nucleic acid amplification methods are isothermal. The polymerase chain reaction (Mullis et al., US Pat. No. 4,683,195; Mullis, US Pat. No. 4,683,202; and Mullis et al., US Pat. USA No. 4,800,159), commonly referred to as PCR, uses multiple cycles of denaturation, renaturation of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target sequence. In a variation called RT-PCR, reverse transcriptase (RT) is used to produce complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA (Gelfand et al., Reverse Transcription with Thermostable DNA Polymerases High Temperature Reverse Transcription, US Pat. Nos. 5,322,770 and 5,310,652). Another method is strand displacement amplification (Walker, G. et al. (1992), Proc. Natl. Acad. Sci. USA 89, 392-396; Walker et al., Nucleic Acid Target Generation, pat. from the USA No. 5,270,184; Walker, Strand Displacement Amplification, pat. U.S. No. 5,455,166; and Walker et al. (1992) Nucleic Acids Research 20, 1691-1696), commonly referred to as SDA, which uses cycles of renaturation of pairs of primer sequences to the opposite strands of a target sequence, prolongation of the primers in the presence of a dNTP to produce a product of double-stranded hemiphosphorothioated primers, endonuclease-mediated nick formation in a hemi-modified restriction endonuclease recognition site, and polymerase-mediated extension of the primers from the 3 'end of the nick to displace the existing strand and produce a strand for the next round of primer renaturation, nicking, and strand displacement, resulting in the geometric amplification of the product. Thermophilic SDA (tSDA) uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (European Pat. No. 0 684 315). Other amplification methods include: nucleic acid sequence-based amplification (Malek et al., US Pat. No. 5,130,238), commonly referred to as NASBA; one that uses an RNA replicase to amplify the probe molecule itself (Lizardi, P. et al. (1988) BioTechnol. 6, 1197-1202), commonly referred to as Qp replicase; a transcription-based amplification method (Kwoh, D. et al. (1989) Proc. Natl. Acad. Sci. USA 86, 11731177); self-sustained sequence replication (Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874-1878; Landgren (1993) Trends in Genetics 9, 199-202; and Lee, H et al., NUCLEIC ACID AMPLIFICATION TECHNOLOGIES (1997)); and transcription-mediated amplification (Kacian et al., Nucleic Acid Sequence Amplification Methods, US Pat. No. 5,480,784; and Kacian et al., US Pat. No. 5,399,491 ), commonly referred to as TMA. For a 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., Eds.), Pp. 51-87 (American Society for Microbiology, Washington, DC). Other exemplary amplification methods suitable for use in accordance with the present invention include rolling circle amplification (RCA) (Lizardi, Rolling Circle Replication Reporter Systems, US Pat. No. 5,854,033); helicase-dependent amplification (HDA) (Kong et al., Helicase Dependent Amplification Nucleic Acids, US Pat. US 2004-0058378 A1); and loop-mediated isothermal amplification (LAMP) (Notomi et al., Process for Synthesizing Nucleic Acid, US Pat. No. 6,410,278).

Preferred transcription-based amplification systems of the present invention include TMA, which employs an RNA polymerase to produce multiple RNA transcripts of a target region (eg, Kacian et al., US Pat. 5,480,784 and 5,399,491; and Becker et al., Single-Primer Nucleic Acid Amplification Methods, US Patent Sol. Pub. No. US 2006-0046265 A1). TMA uses a promoter or promoter-primer oligonucleotide that hybridizes to a target nucleic acid in the presence of a reverse transcriptase and an RNA polymerase to form a double-stranded promoter from which RNA polymerase produces RNA transcripts. These transcripts can be made into templates for the additional rounds of TMA in the presence of a second primer capable of hybridizing to the RNA transcripts. Unlike PCR, LCR or other methods that require thermal denaturation, TMA is an isothermal method that uses an RNase H activity to digest the RNA strand of an RNA: DNA hybrid, thus achieving that the DNA strand is available for hybridization to a primer or a promoter-primer. In general, the provided reverse transcriptase-associated RNase H activity is used for amplification.

In an illustrative TMA method, an amplification primer is a promoter-primer oligonucleotide comprising a promoter sequence that becomes functional when in double-stranded form, located 5 'of a target-binding sequence, that is capable of hybridizing at one site. binding of a target RNA at a location 3 'to the sequence to be amplified. A promoter primer can be referred to as a T7 primer when it is specific for recognition by a T7 RNA polymerase. In certain circumstances, the 3 'end of a promoter-primer, or a subpopulation of such promoter-primers, can be modified to block or reduce the extension of the primers. From an unmodified promoter-primer, reverse transcriptase creates a cDNA copy of the target RNA, while RNase H activity degrades the target RNA. A second amplification primer is then bound to the cDNA. This primer can be referred to as a non-T7 primer to distinguish it from the T7 primer. From this second amplification primer, the

ES 2 358 296 T3 reverse transcriptase creates another strand of DNA, resulting in double-stranded DNA with a functional promoter at one end. When in double-stranded form, the promoter sequence is capable of binding to an RNA polymerase to begin transcription of the target sequence to which it is hybridized in the promoter-primer. An RNA polymerase uses this promoter sequence to produce multiple RNA transcripts (ie, amplicons), generally around 100 to 1,000 copies. Each newly synthesized amplicon can renature with the second amplification primer. Reverse transcriptase can then create a DNA copy, while RNase H activity degrades the RNA of this RNA: DNA double-stranded molecule. The promoter-primer can then be attached to the newly synthesized DNA, enabling the reverse transcriptase to create a double-stranded DNA, from which the RNA polymerase produces multiple amplicons. Thus, a billion-fold isothermal amplification can be carried out by using two amplification primers.

In another illustrative TMA method, one or more features described in Becker et al., Pub. Sun. by pat. from USA US 2006-0046265 A1. Preferred TMA methods in this regard include the use of blocking moieties, termination moieties, and other modifying moieties that provide improved sensitivity and accuracy to the TMA process. Thus, certain preferred embodiments of the present invention employ labeled oligonucleotides, as described herein, in conjunction with the methods described in Becker et al., Pub. Sun. by pat. US No. US 2006-0046265 A1.

Detectable amplification means that a detectable signal associated with an amplification product in an amplification reaction mix rises above a predetermined background or threshold level (end-point amplification) or rises above a background or threshold level. in a predetermined period of time (real-time amplification). See, eg, Light et al., Method for Determining the Amount of an Analyte in a Sample, pub. Sun. by pat. from USA US 20060276972, paragraphs 506-549. The amplification product contains a sequence that has sequence identity to a target nucleic acid sequence or its complement and that can be detected, for example, with an intercalating dye or a detection probe that has specificity towards a region of the sequence. of target nucleic acid or its complement.

Selective Amplification

Selective amplification, as used herein, refers to the amplification of a target nucleic acid sequence according to the present invention wherein the detectable amplification of the target sequence is limited or substantially limited to the amplification of the target sequence. contributed by the sample of interest being analyzed, and is not contributed by the target nucleic acid sequence contributed by some other source in the sample, e.g., contamination present in the reagents or components used during amplification reactions or in the medium or environmental conditions in which the amplification reactions are carried out

Amplification Conditions

"Amplification conditions" mean the conditions that allow the amplification of nucleic acids according to the present invention. Amplification conditions can be, in certain embodiments, less stringent than stringent hybridization conditions described herein. The oligonucleotides used in the amplification reactions of the present invention hybridize to their desired targets under the amplification conditions, but 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. Although the Examples section below provides the preferred amplification conditions for amplifying the target nucleic acid sequences according to the present invention, one of ordinary skill in the art could easily devise other acceptable conditions for carrying out the nucleic acid amplifications. according to the present invention depending on the particular amplification method employed.

Hybridize / Hybridize

Nucleic acid hybridization is the process by which two strands of nucleic acids having completely or partially complementary nucleotide sequences are joined together under predetermined reaction conditions to form a stable double-stranded hybrid. Each nucleic acid strand can be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or the analogs thereof. Thus, hybridization may involve RNA: RNA hybrids, DNA: DNA hybrids, RNA: DNA hybrids, or analogs thereof. The two constituent chains of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. Although these hydrogen bonds are usually formed between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) in single-stranded nucleic acid chains, base pairing can be formed also between bases that are not members of these canonical pairs. Base pairing that is not canonical is well known in the art.

ES 2 358 296 T3 (see, eg, ROGER LP ADAMS ET AL., THE BIOCHEMISTRY OF THE NUCLEIC ACIDS (11th ed., 1992)).

Stringent hybridization conditions or stringent conditions refer to conditions in which a specific detection probe is capable of hybridizing to target nucleic acids over other nucleic acids present in the test sample. It will be appreciated that these conditions may vary depending on factors including the GC content and probe length, the hybridization temperature, the composition of the hybridization reagent or solution, and the degree of hybridization specificity desired. Specific stringent hybridization conditions are provided later in the description.

A nucleic acid hybrid or hybrid or double-stranded molecule means a nucleic acid structure that contains a double-stranded, hydrogen-bonded region, in which each strand is complementary to the other, and in which the region is sufficiently stable under conditions hybridization tests to be detected by means including, but not limited to, chemiluminescent or fluorescent light detection, autoradiography, or gel electrophoresis. Such hybrids can comprise double stranded RNA: RNA, RNA: DNA, or DNA: DNA molecules.

Complementary means that the nucleotide sequences of the similar regions of two single-stranded nucleic acids, or of different regions in the same single-stranded nucleic acid, have a nucleotide base composition that allows the single-stranded regions to hybridize to each other in a double-stranded region, stable with hydrogen bonding under stringent hybridization or amplification conditions. When a contiguous sequence of nucleotides from one single-stranded region is capable of forming a series of hydrogen-bonded canonical base pairs with an analogous nucleotide sequence from the other single-stranded region, such that A pairs with U or T and C is pairs with G, the nucleotide sequences are perfectly complementary.

Preferential hybridization means that under stringent hybridization conditions, certain complementary nucleotide or nucleobase sequences hybridize to form a stable hybrid preferentially over other less stable double-stranded molecules. Non-stably hybridizing means that a stable hybrid does not form in appreciable and / or detectable amounts under a defined set of conditions.

Stable or stably hybridize means that the temperature of a reaction mixture is at least 2 ° C below the melting temperature of a double-stranded nucleic acid molecule.

Promoter Oligonucleotide / Promoter Sequence

As is well known in the art, a promoter is a specific nucleic acid sequence that is recognized by a DNA-dependent RNA polymerase (transcriptase) as a signal to bind nucleic acid and begin RNA transcription at a specific site. For binding, it was generally thought that such transcriptases required DNA that had been made double-stranded in the region comprising the promoter sequence by means of an elongation reaction, however, the present inventors have determined that efficient transcription of the RNA even under conditions where a double-stranded promoter is not formed by an elongation reaction with the template nucleic acid. The template nucleic acid (the sequence to be transcribed) need not be double stranded. Individual DNA-dependent RNA polymerases recognize a variety of different promoter sequences, which can vary markedly in their effectiveness in stimulating transcription. When an RNA polymerase binds to a promoter sequence to initiate transcription, that promoter sequence is not part of the transcribed sequence. Thus, the RNA transcripts produced by it will not include that sequence.

According to the present invention, a promoter oligonucleotide refers to an oligonucleotide comprising first and second regions, and which is preferably modified to prevent the initiation of DNA synthesis from its 3 'end. The first region of a promoter oligonucleotide of the present invention comprises a base sequence that hybridizes to a DNA template, wherein the hybridization sequence is located 3 ', but not necessarily adjacent, to a promoter region. The hybridization portion of a promoter oligonucleotide of the present invention is generally at least 10 nucleotides in length, and can be extended to a length of 15, 20, 25, 30, 35, 40, 50 or more nucleotides. The second region comprises a promoter for an RNA polymerase. A promoter oligonucleotide of the present invention is modified in such a way that it is incapable of being extended by an RNA- or DNA-dependent DNA polymerase, eg, reverse transcriptase, preferably comprising a blocking moiety at its 3 'end as described. described above. Suitable and preferred promoter oligonucleotides are described herein.

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Pan Oligonucleotides / Universal Oligonucleotides

Universal oligonucleotides or pan 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 a class of organisms (such as used herein, the term "class" does not necessarily imply a recognized phylogenetic grouping of organisms). For example, the highly conserved coding sequences of 16S ribosomal RNA contain regions that are found in bacteria, or groupings of bacteria (eg, Eubacteria, gram-positive bacteria, or gram-negative bacteria), but do not exist in humans and other higher organisms. and thus the oligonucleotides can be designed and used in a nucleic acid amplification reaction to detect the presence of bacterial sequences in a sample of interest. See, eg, McCabe et al. (1999) Molecular Genetics and Metabolism 66, 205-211; Schmidt, T. et al. (1994) Meth. Enzymol. 235, 205-222 (method for the identification of pathogens); Kunishima, S. et al., (2000) Transfusion 40, 1420 (method for the detection of bacteria in blood); Greisen, K. (1994) J. Clin. Microbiol. 32, 335-351 (method for the detection of pathogenic bacteria in cerebrospinal fluid); Jordan, J. (2005) J. Mol. Diag. 7, 575-581 (method for the diagnosis of sepsis in neonates); Rothman, R. et al. (2002) J. Infect. Dis. 186, 16771681 (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 have been described for other classes of organisms, such as fungal pathogens. See, eg, Maaroati, Y. et al. (2003) J. Clin. Microbiol. 41, 3293-3298 (method for quantification of Candida albicans in blood); Carr, M. et al. (2005) J. Clin. Microbiol. 43, 3023-3026 (method for the detection of Candida dubliniensis in blood); and White, P. et al. (2003) J. Med. Microbiol. 52, 229-238 (method for the detection of systemic fungal infections). Essentially any known or developed universal oligonucleotide for a given class of organism can be advantageously employed in the methods described herein.

Priming Oligonucleotide

A primer oligonucleotide is an oligonucleotide in which at least the 3 'end is complementary to a nucleic acid template, and which complexes (via hydrogen bonding or hybridization) with the template to provide a primer: template complex. suitable for initiation of synthesis by an RNA- or DNA-dependent DNA polymerase. A primer oligonucleotide is extended by the addition of nucleotide bases covalently attached at its 3 'end, the bases of which are complementary to the template. The result is a primer extension product. A primer oligonucleotide of the present invention is generally at least 10 nucleotides in length, and can be extended to a length of 15, 20, 25, 30, 35, 40, 50 or more nucleotides. Suitable and preferred primer oligonucleotides are described herein. Virtually all known DNA polymerases (including reverse transcriptases) require complexation of an oligonucleotide to a single-stranded template (priming) to initiate DNA synthesis, while RNA replication and transcription (RNA copy from DNA) generally does not require a primer. By its very nature of being elongated by a DNA polymerase, a primer oligonucleotide does not comprise a 3 'blocking moiety.

Displacer Oligonucleotide

A displacer oligonucleotide is a primer oligonucleotide that hybridizes to a template nucleic acid 5 'to a near primer oligonucleotide hybridized to the 3' end of a target sequence (referred to herein as direct-priming oligonucleotide). In position 5 'it means that a 3' end of the displacer oligonucleotide complexes with the template nucleic acid 5 'to a 3' end of the forward primer oligonucleotide. When hybridized to the template nucleic acid, the 3'-terminal base of the displacer oligonucleotide is preferably adjacent to, or spaced from, the 5-terminal base of the forward primer oligonucleotide. More preferably, the 3'-terminal base of the displacer oligonucleotide is spaced 5 to 35 bases apart from the 5'-terminal base of the forward primer oligonucleotide. The displacer oligonucleotide can be provided to the reaction mixture at the same time as the forward primer oligonucleotide or after the forward primer oligonucleotide has had sufficient time to hybridize to the template nucleic acid. The elongation of the forward priming oligonucleotide can be initiated before or after providing the displacer oligonucleotide to the reaction mixture. Under amplification conditions, the displacer oligonucleotide is elongated in a template-dependent manner, thereby displacing the primer elongation product comprising the forward-priming oligonucleotide that is complexed with the template nucleic acid. Once displaced from the template nucleic acid, the primer extension product comprising the forward primer oligonucleotide is available for complexation with the promoter oligonucleotide. The forward priming oligonucleotide and the displacer oligonucleotide preferentially hybridize to the target nucleic acid. Examples of displacer oligonucleotides and their uses are described in Becker et al., Methods and Kits for Amplifying DNA, sol. by pat. No. 11 / 681.104, which enjoys common ownership herewith.

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Blocking Rest

As used herein, a blocking moiety is a substance used to block the 3 'end of an oligonucleotide or other nucleic acid so that it cannot be efficiently extended by a nucleic acid polymerase. A blocking moiety may be a small molecule, eg, a phosphate or ammonium group, or it may be a modified nucleotide, eg, a 3'2 'dideoxynucleotide or 3'-deoxyadenosine 5'-triphosphate (cordycepin ), or other modified nucleotide. Additional blocking moieties include, for example, the use of a nucleotide or a short nucleotide sequence that has 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, eg, Arnold et al., Non-Nucleotide Linking Reagents for Nucleotide Probes, US Pat. No. 6,031,091), phosphorothioate, alkane diol residues, peptide nucleic acid (PNA), nucleotide residues lacking a 3 'hydroxyl group at the 3' end, or a nucleic acid binding protein. Preferably, the 3 'blocking moiety comprises a nucleotide or nucleotide sequence having a 3'to-5' orientation or a 3 'non-nucleotide moiety, and not a 3'2'-dideoxynucleotide or a 3' end that it has a free hydroxyl group. Other methods of preparing 3 'blocking oligonucleotides are well known to those of ordinary skill in the art.

Binding molecule

As used herein, a binding molecule is a substance that hybridizes to or otherwise binds to an RNA target nucleic acid adjacent or near the 5 'end of the desired target sequence, to limit a primer extension product of DNA to a desired length, ie, a primer extension product having a generally defined 3 'end. As used herein, the phrase "defined 3 'end" means that the 3' end of a primer extension product is not completely indeterminate, as would be the case in a primer extension reaction occurring in the absence of a molecule. rather, the 3 'end of the primer extension product is generally known in a small range of bases. In certain embodiments, a binding molecule comprises a region of bases. The base region can be DNA, RNA, a DNA: RNA chimeric molecule, or an analog thereof. Binding molecules that comprise a base region can be modified in one or more ways, as described herein. Exemplary base regions include stopper and digest oligonucleotides, as described below. In other embodiments, a binding molecule can comprise, for example, a protein or drug capable of binding RNA with sufficient affinity and specificity to limit a DNA primer extension product to a predetermined length.

Oligonucleotide Termination

In the present invention, a terminating oligonucleotide is an oligonucleotide comprising a sequence of bases that is complementary to a region of the target nucleic acid near the 5 'end of the target sequence, to terminate the primer extension of a nascent nucleic acid that includes a priming oligonucleotide, whereby a defined 3 'end is provided for the nascent nucleic acid strand. A terminator oligonucleotide is designed to hybridize to the target nucleic acid in a position sufficient to achieve the desired 3 'end for the nascent nucleic acid strand. The positioning of the terminator oligonucleotide is flexible depending on its design. A terminator oligonucleotide can be modified or unmodified. In certain embodiments, the terminator oligonucleotides are synthesized with at least one or more 2'-O-ME ribonucleotides. These modified nucleotides have been shown to have superior thermal stability in complementary double-stranded molecules. 2'-O-ME ribonucleotides also function to increase the resistance of oligonucleotides to exonucleases, thereby increasing the half-life of modified oligonucleotides. See, eg, Majlessi et al. (1988) Nucleic Acids Res. 26, 2224-9. Other modifications as described elsewhere herein may be used in addition to or in place of the 2'-O-ME ribonucleotides. For example, a terminating oligonucleotide can comprise PNA or an LNA. See, eg, Petersen et al. (2000) J. Mol. Recognit. 13, 44-53. A terminator oligonucleotide of the present invention generally includes a blocking moiety at its 3 'end to prevent elongation. A terminating oligonucleotide may also comprise a protein or peptide linked to the oligonucleotide to terminate further extension of a nascent nucleic acid strand by a polymerase. A terminator oligonucleotide of the present invention is generally at least 10 bases in length, and can be extended to a length of 15, 20, 25, 30, 35, 40, 50 or more nucleotides. Suitable and preferred termination oligonucleotides are described herein. It should be noted that although a terminating oligonucleotide generally or necessarily includes a 3 'blocking moiety, the 3' blocked oligonucleotides are not necessarily terminating oligonucleotides. Other oligonucleotides of the present invention, eg, promoter oligonucleotides and protective oligonucleotides are also generally or necessarily 3 'blocked.

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Insertion Sequence

As used herein, an insertion sequence is a sequence positioned between the first region (ie, the template-binding portion) and the second region of a promoter oligonucleotide. The insertion sequences are preferably 5 to 20 nucleotides in length, more preferably 6 to 18 nucleotides, and most preferably 6 to 12 nucleotides. The inclusion of insertion sequences in the promoter oligonucleotides increases the rate at which RNA amplification products are formed. Exemplary insertion sequences are described herein.

Target Capture

Target capture, as used herein, includes any technique effective to remove all or substantially all of the unhybridized tagged oligonucleotide after hybridization of the tagged oligonucleotide to a target nucleic acid sequence, but prior to amplification of the target nucleic acid sequence. target nucleic acid sequence. In general, target capture involves capturing a target polynucleotide on a solid support, such as magnetically attractable particles, where the solid support retains the target polynucleotide during one or more wash steps of the target polynucleotide purification process. In this manner, a target polynucleotide is substantially purified from the unhybridized labeled oligonucleotide prior to a subsequent nucleic acid amplification step. Numerous target capture methods are known, and are suitable for use in conjunction with the methods described herein.

For example, an illustrative approach described in pub. Sun. by pat. US 2006-0068417 A1 uses at least one capture probe oligonucleotide containing a region complementary to the target and a member of a specific binding pair that binds the target nucleic acid to a probe immobilized on a support capture, so a capture hybrid is formed that is separated from the other components of the sample. In another illustrative method, Weisburg et al., In pat. No. 6,110,678, describes a method of capturing a target polynucleotide in a sample on a solid support, such as magnetically attractable particles, with an immobilized probe attached by using a capture probe and two conditions of different hybridization, preferably differing only in temperature. The two hybridization conditions control the order of hybridization, in which the first hybridization conditions allow hybridization of the capture probe to the target polynucleotide, and the second hybridization conditions allow hybridization of the capture probe to the immobilized probe. . The method can be used to detect the presence of a target polynucleotide in a sample by detecting the captured target polynucleotide or the amplified target polynucleotide.

Another illustrative target capture technique involves a sandwich hybridization technique to capture and detect the presence of a target polynucleotide. See Ranki et al., Detection of Microbial Nucleic Acids By a One-Step Sandwich Hybridization Test, pat. U.S. No. 4,486,539. The technique involves capturing the target polynucleotide by a probe attached to a solid support and hybridizing a detection probe to the captured target polynucleotide. Detection probes not hybridized to the target polynucleotide are easily removed by washing the solid support. Thus, the remaining marker is associated with the target polynucleotide initially present in the sample.

Another illustrative target capture technique involves a method using a mediator polynucleotide that hybridizes to both the target polynucleotide and the polynucleotide fixed on a solid support. See Stabinsky, Methods and Kits for Performing Nucleic Acid Hybridization Assays, pat. U.S. No. 4,751,177. The mediator polynucleotide binds the target polynucleotide to the solid support to produce a bound target. A labeled probe can hybridize to the bound target, and unbound labeled probe can be washed off the solid support.

Still another illustrative target capture technique is described in Englelhardt, Capture Sandwich Hybridization Method and Composition, pat. from the USA No. 5,288,609, which describes a method for detecting a target polynucleotide. The method uses two single-stranded polynucleotide segments complementary to the same or opposite strands of the target, and results in the formation of a double-stranded hybrid with the target polynucleotide. In one embodiment, the hybrid is captured on a support.

In another illustrative target capture technique, nucleic acid detection methods and kits use oligonucleotide primers labeled with specific binding molecules to immobilize the primers and primer extension products. See Burdick et al., Diagnostic Kit and Method Using a Solid Phase Capture Means for Detecting Nucleic Acids, sol. by pat. European n ° 0 370 694 A2. The marker complexes specifically with its receptor, which is attached to a solid support.

The above capture techniques are illustrative only, and are not limiting. In fact, virtually any method available to the skilled technician can be used as long as it is effective.

ES 2 358 296 T3 to remove all or substantially all of the unhybridized labeled oligonucleotide after hybridization of the labeled oligonucleotide to a target nucleic acid sequence, but prior to amplification of the target nucleic acid sequence, as described herein .

Probe

Probe or detection probe means a molecule comprising an oligonucleotide that has a base sequence partially or completely complementary to a region of a target sequence to be detected, such that it hybridizes to it under stringent hybridization conditions. As one of ordinary skill in the art would understand, a probe comprises an isolated nucleic acid molecule, or an analog thereof, in a form not found in nature without human intervention (e.g., recombined with an exogenous nucleic acid, isolated, or purified to some extent).

The probes of this invention may have additional nucleosides or nucleobases outside of the targeted region as long as such nucleosides or nucleobases do not substantially affect hybridization under stringent hybridization conditions and, in the case of detection probes, do not prevent preferential hybridization to the target nucleic acid. A non-complementary sequence can also be included, such as a target capture sequence (generally a stretch of homopolymer, such as a poly-A, poly-T, or poly-U tail), a promoter sequence, a binding for RNA transcription, a recognition site for a restriction endonuclease, or may contain sequences that will confer a desired secondary or tertiary structure, such as a catalytic active site or hairpin structure on the probe, in the target nucleic acid, or both.

The probes preferably include at least one detectable label. The label can be any suitable labeling substance, including, but not limited to, a radioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye, a hapten, a chemiluminescent molecule, a fluorescent molecule, a phosphorescent molecule, a electrochemiluminescent molecule, a chromophore, a region of a base sequence that is incapable of stably hybridizing to the target nucleic acid under the indicated conditions, and mixtures of these. In a particularly preferred embodiment, the label is an acridinium ester. The probes can also include interacting markers that emit different signals depending on whether the probes have hybridized to the target sequences. Examples of interacting labels include enzyme / substrates, enzyme / cofactor, luminescent / quencher molecule, luminescent / adduct molecule, dye dimers, and Forrester energy transfer pairs. Certain probes of the present invention do not include a marker. For example, unlabeled capture probes can be used to enrich for target sequences or replicates thereof, which can then be detected by a second detection probe. See, eg, Weisburg et al., Pat. U.S. No. 6,534,273. Although detection probes are generally labeled, certain detection techniques do not require the probe to be labeled. See, eg, Nygren et al., Devices and Methods for Optical Detection of Nucleic Acid Hybridization, pat. U.S. No. 6,060,237.

Stable or stable for detection means that the temperature of a reaction mixture is at least 2 ° C below the melting temperature of a double-stranded nucleic acid. The temperature of the reaction mixture is preferably at least 5 ° C below the melting temperature of the double-stranded nucleic acid, and even more preferably at least 10 ° C below the melting temperature of the reaction mixture.

Preferentially hybridizing means that under stringent hybridization conditions, the probes of the present invention hybridize to their target sequences, or replicates thereof, to form stable probe: target hybrids, while at the same time minimizing the formation of probe: no hybrids. target. Thus, a probe hybridizes to a target sequence or replicates thereof to a point sufficiently greater than a non-target sequence to allow a person of ordinary skill in the art to accurately quantify the replicates of RNA or complementary DNA. (cDNA) of the target sequence formed during amplification.

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

Nucleic Acid Identity

In certain embodiments, a nucleic acid of the present invention comprises a contiguous base region that is at least 80%, 90%, or 100% identical to a contiguous base region of a reference nucleic acid. For short nucleic acids, eg, certain oligonucleotides of the present invention, the degree of identity between a base region of a test nucleic acid and a base region of a reference nucleic acid can be determined by manual alignment.

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Identity is determined by comparing only the sequence of nitrogenous bases, regardless of the carbohydrate and backbone regions of the nucleic acids being compared. Thus, the reference base sequence alignment can be DNA: DNA, RNA: RNA, DNA: RNA, RNA: DNA, or any combination or analogs thereof. Equivalent RNA and DNA base sequences can be compared by converting U (in RNA) to T (in DNA).

Mold

A template is a nucleic acid molecule that is to be copied by a nucleic acid polymerase. A template can be single-stranded, double-stranded, or partially double-stranded, depending on the polymerase. The synthesized copy is complementary to the template or at least one strand of a double or partially double stranded template. Both RNA and DNA are generally synthesized in the 5'-to-3 'direction, and the two strands of a double-stranded nucleic acid align so that the 5' ends of the two strands are at opposite ends of the double-stranded molecule (and, necessarily, so are the 3 'ends). Although according to the present invention, a target sequence is always a template, templates can also include secondary primer extension products and amplification products.

DNA Dependent DNA Polymerase

A DNA-dependent DNA polymerase is an enzyme that synthesizes a complementary DNA copy from a DNA template. Examples are Taq DNA polymerase, a very 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 polymerases from bacteriophage T4, Phi-29, M2, or T5. The DNA-dependent DNA polymerases of the present invention can be natural enzymes isolated from bacteria or bacteriophages or expressed recombinantly, or they can be modified or evolved forms that have been modified to possess certain desirable characteristics, eg, thermostability, or the ability to recognize or synthesize a DNA strand from various modified templates. All known DNA-dependent DNA polymerases require a complementary primer to initiate synthesis. It is known that under suitable conditions a DNA-dependent DNA polymerase can synthesize a complementary DNA copy from an RNA template. RNA-dependent DNA polymerases (described below) also generally have DNA-dependent DNA polymerase activity. An example of such a polymerase is MasterAmp ™ Tth DNA polymerase, which has both DNA-dependent and RNA-dependent DNA polymerase activity (i.e., reverse transcriptase) that can be used in PCR and RT-PCR amplification reactions (Epicenter Biotechnologies, Madison, WI).

DNA Dependent RNA Polymerase (Transcriptase)

A DNA-dependent RNA polymerase or transcriptase is an enzyme that synthesizes multiple copies of RNA from a double-stranded or partially double-stranded DNA molecule having a promoter sequence that is normally double-stranded. RNA molecules (transcripts) are synthesized in the 5'-to-3 'direction starting at a specific position just 3' from the promoter. Examples of transcriptases are the DNA-dependent RNA polymerase of E. coli and bacteriophages T7, T3, and SP6.

RNA Dependent DNA Polymerase (Reverse Transcriptase)

An RNA-dependent DNA polymerase or reverse transcriptase (RT) is an enzyme that synthesizes a complementary DNA copy from an RNA template. All known reverse transcriptases also have the ability to produce a complementary DNA copy from a DNA template; thus, they are DNA polymerases dependent on both DNA and RNA. RTs can also have RNase H activity. Moloney murine leukemia virus derived reverse transcriptase (MMLV-RT) is preferred. A primer is necessary to initiate synthesis with the RNA and DNA templates.

Selective RNases

As used herein, a selective RNase is an enzyme that degrades the RNA portion of a double-stranded RNA: DNA molecule but not the single-stranded RNA, double-stranded aRn, or DNA. An exemplary selective RNase is RNase H. Enzymes other than RNase H that possess the same or similar activity are also contemplated by the present invention. Selective RNases can be endonucleases or exonucleases. Most reverse transcriptase enzymes contain RNase H activity in addition to their polymerase activities. However, other sources of RNase H are available without an associated polymerase activity. Degradation can result in the separation of RNA from an RNA: DNA complex. Alternatively, a selective RNase can simply cut RNA at various positions such that portions of the RNA are denatured or allow enzymes to unwind portions of the RNA.

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Other enzymes that selectively degrade target RNA sequences or RNA products of the present invention will be readily apparent to those of ordinary skill in the art.

Sense / Antisense String (s)

Discussions of nucleic acid synthesis are greatly simplified and clarified by adopting terms to name the two complementary strands of a double-stranded nucleic acid molecule. Traditionally, the strand that encodes the sequences used to produce structural proteins or RNAs is called the sense strand (+) and its complement strand antisense.Now it is known that in many cases both strands are functional, and the assignment of the sense name to one and antisense the other must be arbitrary. However, the terms are very useful in denoting nucleic acid sequence orientation, and will be used herein for that purpose.

System Specificity

The term specificity, in the context of an amplification system, is used herein to refer to the characteristic of an amplification system that describes its ability to distinguish between target sequences and non-target sequences depending on the sequence and on the test conditions. In terms of nucleic acid amplification, specificity generally refers to the ratio of the number of specific amplicons produced to the number of secondary products (i.e., the ratio of signal to noise), described in more detail below. .

Sensitivity

The term "sensitivity" is used herein to refer to the precision with which a nucleic acid amplification reaction can be detected or quantified. The sensitivity of an amplification reaction is generally a measure of the smallest copy number of the target nucleic acid that can be reliably detected in the amplification system, and will depend, for example, on the detection assay being employed, and of the specificity of the amplification reaction, that is, the ratio of specific amplicons to by-products.

Bioprocess

A bioprocess, as used herein, refers generally to any process in which living cells, or components thereof, are intentionally or unintentionally present. For example, essentially any manufacturing or other process that employs one or more samples or sample streams, at least one of which comprises living cells, or the components thereof, or which may comprise such cells or components as a result from unintentional contamination, it 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 living cells or components thereof 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 samples or bioprocess streams. Furthermore, purification / sterilization requirements on certain samples / streams of a bioprocess can be advantageously reduced by using the methods of the invention as set forth herein.

*****

As discussed above, the present invention is generally directed to nucleic acid amplification methods that desirably reduce or eliminate false positive amplification signals that result from contaminating biological material, such as nucleic acid material, which may be present in one or more reagents, samples or components that are used in an amplification reaction, or they may be present in the medium in which the amplification reactions are carried out. The invention further offers the advantage of requiring less stringent purification and / or sterility efforts than is conventionally necessary to ensure that the enzymes and other reagents used in the amplification reactions, and the environment in which the amplification reactions are carried out out, they are free from contamination by bacterial and other nucleic acids that can produce false positive results. Therefore, the methods of the invention are especially useful for detecting, monitoring and / or quantifying microorganisms (or contaminating nucleic acids from other sources) in clinical samples, bioprocess samples or in sample streams, food, water, industrial samples and environment, seed reserves, and other types of materials in which it may be necessary to detect and / or monitor the presence of microorganisms or other forms of contamination.

The present invention can be adapted for use in virtually any amplification procedure that requires a template-binding primer oligonucleotide capable of being elongated.

ES 2 358 296 T3 presence of a nucleic acid polymerase. Incorporation of the labeled oligonucleotides (or heterologous marker sequences) into such primer-dependent amplification procedures can be carried out without substantially modifying the reagents and reaction conditions of such procedures. Any necessary modifications should be minor, and would be within the knowledge and ability of a skilled molecular biologist. The following are descriptions of various illustrative amplification procedures that the labeled oligonucleotides adopt.

FIG. 1 illustrates an adaptation of a transcription-based, isothermal amplification reaction known as reverse transcription-mediated amplification (rTMA), various aspects of which are described in Becker et al., Pub. Sun. by pat. US No. US 2006-0046265 A1. The reaction of this illustrative embodiment is initiated by treating an RNA target sequence from a nucleic acid sample with a labeled primer oligonucleotide and a terminator oligonucleotide. The labeled primer oligonucleotide includes a target hybridizing sequence that hybridizes to a 3 'end of the target sequence and a marker sequence located 5' to the target hybridizing sequence. The terminator oligonucleotide hybridizes to a target nucleic acid containing the target sequence near the 5 'end of the target sequence. The terminator oligonucleotide is used to terminate the primer extension of a nascent nucleic acid that includes the labeled primer oligonucleotide. Thus, the target nucleic acid forms a stable complex with the primer oligonucleotide labeled at the 3 'end of the target sequence and the terminator oligonucleotide located adjacent to or close to the 5' end of the target sequence before initiating a reaction reaction. primer extension. See FIG. 1, Step 1. The unhybridized labeled primer oligonucleotide is made unavailable for hybridization to the target sequence before initiating a primer extension reaction with the labeled primer oligonucleotide, preferably by inactivating and / or removing the oligonucleotide from unhybridized labeled priming of the nucleic acid sample.

An extension reaction is then initiated from the 3 'end of the primer oligonucleotide labeled with a DNA polymerase, eg, reverse transcriptase, to produce a first DNA primer extension product that includes the marker sequence and a region complementary to the target sequence. See FIG. 1, Stages 2 and 3. The first DNA primer extension product is then separated from the target sequence by using an enzyme that selectively degrades the target sequence (eg, RNase H activity). See FIG. 1, Stage 4.

The first DNA primer extension product is then treated with a promoter oligonucleotide having a hybridization sequence and a promoter for an RNA polymerase located 5 'to the hybridization sequence. The hybridization sequence hybridizes to a region of the first DNA primer extension product that is complementary to the 3 'end of the target sequence, thereby forming a promoter oligonucleotide: first DNA primer extension product hybrid. 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 (eg, a nucleotide sequence that has a 3'-orientation). to 5'). See FIG. 1, Stage 5. The 3 'end of the first DNA primer extension product is preferably extended to add a complementary sequence to the promoter, resulting in the formation of a double-stranded promoter sequence. See FIG. 1, Stages 6 and 7. Multiple copies of a first RNA product complementary to at least a portion of the first DNA primer extension product, which does not include the promoter portion, are then transcribed using an RNA polymerase that recognizes the double-stranded promoter and initiates the transcript from him. See FIG. 1, Stages 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 marker sequence.

The first RNA products are treated with a primer oligonucleotide that hybridizes to the complement of the marker sequence to form a primer oligonucleotide: first RNA product hybrid, and the 3 'end of the primer oligonucleotide is elongated with DNA polymerase to produce a second DNA primer extension product complementary to the first RNA product. See FIG. 1, Stages 10-12. The second DNA primer extension product is then separated from the first RNA product by use of an enzyme that selectively degrades the first RNA product (eg, RNase H activity). See FIG. 1, Stage 13.

The second DNA primer extension product is treated with the promoter oligonucleotide, which hybridizes to the 3 'end of the second DNA primer extension product to form a promoter oligonucleotide: second DNA primer extension product hybrid. See FIG. 1, Step 14. The promoter oligonucleotide: second DNA primer extension product hybrid then re-enters the amplification cycle in Step 6 of FIG. 1, in which transcription is initiated from the double-stranded promoter and the cycle continues.

FIG. 3 illustrates an adaptation of a transcription-based, isothermal amplification reaction, termed transcription-mediated amplification (TMA), various aspects of which are described in Kacian et al., Pat. U.S. No. 5,399,491 and 5,824,518. The reaction of this realization

Illustrative ES 2 358 296 T3 begins by treating an RNA target sequence from a nucleic acid sample with a labeled promoter oligonucleotide. The tagged promoter oligonucleotide includes a tag sequence, a target hybridization sequence, and a promoter sequence for an RNA polymerase, wherein the target hybridization sequence hybridizes to a 3 'end of the target sequence. Thus, the target sequence forms a stable complex with the promoter oligonucleotide labeled at the 3 'end of the target sequence before initiating a primer extension reaction. See FIG. 3, Step 1. The promoter sequence is located 5 'to the marker sequence, and the marker sequence is located 5' to the target hybridization sequence. The unhybridized tagged promoter oligonucleotide is made unavailable for hybridization to the target sequence before initiating a primer extension reaction with the tagged primer oligonucleotide, preferably by inactivating and / or removing the unhybridized tagged primer oligonucleotide from the nucleic acid sample.

An extension reaction is then initiated from the 3 'end of the promoter oligonucleotide labeled with a DNA polymerase, eg, reverse transcriptase, to produce a first DNA primer extension product that includes the marker sequence and the promoter sequence. and a region complementary to the target sequence. See FIG. 1, Stages 2 and 3. The first DNA primer extension product is then separated from the target sequence to which it is hybridized by using an enzyme that selectively degrades that part of the target sequence that hybridizes to the first DNA primer extension product ( eg, RNase H activity). See FIG. 3, Stage 4.

The first DNA primer extension product is then treated with a primer oligonucleotide that hybridizes to a region of the first DNA primer extension product that is complementary to a 5 'end of the target sequence, thereby forms a primer oligonucleotide: first DNA primer extension product. See FIG. 3, Stage 5. The 3 'end of the primer oligonucleotide is extended by a DNA polymerase to produce a second DNA primer extension product complementary to at least a portion of the first DNA primer extension product, and containing a double-stranded promoter sequence. See FIG. 3, Stages 6 and 7. This second DNA primer extension product is used as a template to transcribe multiple copies of a first RNA product complementary to the second DNA primer extension product, which does not include the promoter portion, by using an RNA polymerase that it recognizes the double-stranded promoter and initiates transcription from it. See FIG. 3, Stages 8 and 9. The base sequence of the first RNA product is substantially identical to the base sequence of the marker sequence and to the complement of the target sequence.

The first RNA product is treated with the primer oligonucleotide, the 3 'end of which is extended by DNA polymerase to produce a third DNA primer extension product complementary to the first RNA product. See FIG. 3, Stages 10-12. The third DNA primer extension product is separated after the first RNA product by use of an enzyme that selectively degrades the first RNA product (eg, RNAse H activity). See FIG. 3, Step 13. The third DNA primer extension product is treated with a promoter oligonucleotide having a hybridization sequence that hybridizes to a complement of the marker sequence at the 3 'end of the third DNA primer extension product, and further comprises a promoter for an RNA polymerase that is located 5 'to the hybridization sequence. See FIG. 3, Stage 14. The 3 'end of the third DNA primer extension product is extended to add a sequence complementary to the promoter sequence. See FIG. 3, Step 15. The 3 'end of the promoter oligonucleotide is extended with DNA polymerase to produce a fourth DNA primer extension product complementary to the third DNA primer extension product. See FIG. 3, Stage 16. Multiple copies of a second RNA product complementary to the third DNA primer extension product, which do not include the promoter portion, 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 marker sequence and to the complement of the target sequence.

FIG. 5 illustrates an adaptation of an rTMA amplification reaction to amplify a target DNA sequence, various aspects of which are described in Becker et al., Sol. by pat. U.S. Serial No. 11 / 681.104. The reaction of this illustrative embodiment is initiated by treating a target DNA sequence of a nucleic acid sample with a labeled primer oligonucleotide and a terminator oligonucleotide. The labeled primer oligonucleotide includes a target hybridization sequence hybridized to a 3 'end of the target sequence and a marker sequence located 5' of the target hybridization sequence. The target hybridization sequence preferably hybridizes to a single-stranded form of the target sequence, although it can hybridize to a double-stranded form of the target sequence via strand invasion, which can be facilitated, for example, by local partial denaturation. of DNA (eg, AT-rich regions), low salinity conditions, and / or the use of DMSO and / or osmolytes, such as betaine. The target sequence is preferably made single-stranded by heating the nucleic acid sample. The terminator oligonucleotide hybridizes to a region of a target nucleic acid that contains the target sequence nearby

ES 2 358 296 T3 of the 5 'end of the target sequence. The terminator oligonucleotide is used to terminate the primer extension of a nascent nucleic acid that includes the labeled primer oligonucleotide. Thus, the target nucleic acid forms a stable complex with the primer oligonucleotide labeled at the 3 'end of the target sequence and the terminator oligonucleotide located adjacent or close to the 5' end of the target sequence. See FIG. 5, Stages 1-3. The unhybridized labeled primer oligonucleotide is made unavailable for hybridization to the target sequence prior to initiating a primer extension reaction with the labeled primer oligonucleotide, preferably by inactivating and / or removing the unhybridized labeled primer oligonucleotide from the nucleic acid sample.

An extension reaction is then initiated from the 3 'end of the primer oligonucleotide labeled with a DNA polymerase, eg, reverse transcriptase, to produce a first DNA primer extension product that includes the marker sequence and a region complementary to the target sequence. See FIG. 5, Stages 4 and 5.

The nucleic acid sample is subsequently treated with a displacer oligonucleotide that hybridizes to the target nucleic acid in position 5 'of the labeled oligonucleotide so that a primer extension reaction can be initiated from it, so that the first extension product Primer of DNA is displaced when the 3 'end of the displacer oligonucleotide is elongated by DNA polymerase. See FIG. 5, Stages 6-8. The order of steps illustrated is not intended to imply that the nucleic acid sample of this embodiment must be treated with the labeled primer oligonucleotide before it is treated with the displacer oligonucleotide to be operational. In certain embodiments, it is preferable to cause these two oligonucleotides to hybridize to the target nucleic acid in a substantially simultaneous manner.

The first DNA primer extension product is then treated with a promoter oligonucleotide having a hybridization sequence and a promoter for an RNA polymerase located 5 'to the hybridization sequence. The hybridization sequence hybridizes to a region of the first DNA primer extension product that is complementary to the 3 'end of the target sequence, thereby forming a promoter oligonucleotide: first DNA primer extension product hybrid. 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 (eg, a nucleotide sequence that has a 3'-a- orientation). 5'). See FIG. 5, Step 9. The 3 'end of the first DNA primer extension product is extended to add complementary sequences to the promoter, resulting in the formation of a double-stranded promoter sequence. See FIG. 5, Stages 10 and 11. Multiple copies of a first RNA product complementary to at least a portion of the first DNA primer extension product, which do not include the promoter, are transcribed using a promoter-recognizing RNA polymerase double-stranded and starts transcription from it. See FIG. 5, Stage 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 marker sequence.

The first RNA products are contacted with a primer oligonucleotide that hybridizes to the complement of the marker sequence to form a primer oligonucleotide: first RNA product hybrid, and the 3 'end of the primer oligonucleotide is elongated with DNA polymerase to produce a second DNA primer extension product complementary to the first RNA product. See FIG. 5, Stage 14-16. The second DNA primer extension product is separated from the first RNA product by use of an enzyme that selectively degrades the first RNA product (eg, RNase H activity). See FIG. 5, Stage 17.

The second DNA primer extension product is treated with the promoter oligonucleotide to form a promoter oligonucleotide: second DNA primer extension product hybrid. See FIG. 5, Step 18. The promoter oligonucleotide: second primer extension product hybrid then re-enters the amplification cycle in Step 10 of FIG. 5, in which transcription is initiated from the double-stranded promoter and the cycle continues.

FIG. 7 illustrates an adaptation of a polymerase chain reaction (PCR), various aspects of which are described, for example, in Mullis et al., Pat. US Nos. 4,683,195 and 4,800,159; Mullis, pat. US No. 4,682,202; and Gelfand et al., pat. U.S. No. 5,804,375. The reaction of this illustrative embodiment is initiated by treating a denatured DNA target sequence from a nucleic acid sample with a labeled primer oligonucleotide. The labeled primer oligonucleotide includes a target hybridizing sequence that hybridizes to a 3 'end of the target sequence and a marker sequence located 5' to the target hybridizing sequence. Thus, the target sequence forms a stable complex with the labeled primer oligonucleotide at the 3 'end of the target sequence before initiating a primer extension reaction. See FIG. 7, Stages 1-3. The unhybridized labeled primer oligonucleotide is made unavailable for hybridization to the target sequence prior to initiating a primer extension reaction with the oligonucleotide of

ES 2 358 296 T3 labeled primer, preferably by inactivating and / or removing the unhybridized labeled primer oligonucleotide from the nucleic acid sample.

An extension reaction is then initiated from the 3 'end of the primer oligonucleotide labeled with a DNA polymerase, e.g., Taq DNA polymerase, to produce a first DNA primer extension product that includes the marker sequence and a region complementary to the target sequence. See FIG. 7, Stages 4 and 5. Next, the double-stranded product resulting from the first primer extension reaction is denatured, and the first DNA primer extension product is contacted with a first primer oligonucleotide that hybridizes to a region of the first primer extension product. DNA that is complementary to the 5 'end of the target sequence. See FIG. 7, Stages 6 and 7.

In a second primer extension reaction, the 3 'end of the first priming oligonucleotide is extended with DNA polymerase to produce a second DNA primer extension product that is complementary to a part of the first primer extension product and that includes the target sequence and the complement of the marker sequence. See FIG. 7, Stages 8 and 9. The double-stranded product resulting from the second primer extension reaction is denatured, and the second DNA primer extension product is contacted with a second primer oligonucleotide that hybridizes to the complement of the marker sequence. See FIG. 7, Stages 10 and 11.

The 3 'end of the second priming oligonucleotide is then extended in a third primer extension reaction with DNA polymerase to produce a third DNA primer extension product that is complementary to the second DNA primer extension product. See 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 for participation in repeated cycles of a polymerase chain reaction by use as primers. of the first and second priming oligonucleotides. See FIG. 7, Stages 14-16.

FIG. 9 illustrates an adaptation of a reverse transcription polymerase chain reaction (RT-PCR), various aspects of which are described, for example, in Gelfand et. al., pat. US Nos. 5,322,770 and 5,310,652. The reaction of this illustrative embodiment is initiated by treating an RNA target sequence in a nucleic acid sample with a labeled primer oligonucleotide. The labeled primer oligonucleotide includes a target hybridization sequence and a marker sequence located 5 'to the target hybridization sequence. Thus, the target sequence forms a stable complex with the labeled primer oligonucleotide at the 3 'end of the target sequence before initiating a primer extension reaction. See FIG. 9, Stage 1. The unhybridized labeled primer oligonucleotide is made unavailable for hybridization to the target sequence prior to initiating a primer extension reaction with the labeled primer oligonucleotide, preferably by inactivating and / or removing the unhybridized labeled primer oligonucleotide from the nucleic acid sample.

An extension reaction is then initiated from the 3 'end of the primer oligonucleotide labeled with a DNA polymerase, eg, MasterAmp ™ Tth DNA polymerase, to produce a first DNA primer extension product including the marker 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 to which it hybridizes by using an enzyme that selectively degrades that part of the target nucleic acid that contains the target sequence that is complementary to the first product. of DNA primer extension (eg, RNase H activity). See FIG. 9, Stage 4.

Next, the first DNA primer extension product is treated with a first primer oligonucleotide that hybridizes to a region of the first DNA primer extension product that is complementary to the 5 'end of the target sequence to form a primer hybrid. DNA primer extension product: primer oligonucleotide. See FIG. 9, Stage 5. A second primer extension reaction extends the 3 'end of the first priming oligonucleotide with DNA polymerase to produce a second DNA primer extension product complementary to at least a portion of the first primer extension product and includes the target sequence and the complement of the marker sequence. See FIG. 9, Stages 6 and 7. The first and second DNA primer extension products are then separated from each other by denaturation. See FIG. 9, Step 8. The first and second extension products are then available to participate in repeated cycles of a polymerase chain reaction by using as primers the first primer oligonucleotide and a second primer oligonucleotide that hybridizes to complement. of the marker sequence. See FIG. 9, Stages 9 and 10; FIG. 7, Stages 13-16.

ES 2 358 296 T3

In other illustrative embodiments of the present disclosure, a heterologous marker sequence that has not been part of a tagged target nucleic acid sequence is inactivated prior to exposing the tagged target nucleic acid sequence to reagents and conditions sufficient for detectable amplification of a sequence. of target nucleic acid. In a preferred aspect, the inactivated heterologous marker sequence is in the form of a labeled oligonucleotide that did not hybridize to the target nucleic acid sequence. Labeled oligonucleotides are described above, and include first and second regions, in which the first region comprises a target hybridizing sequence that hybridizes to a 3 'end of a target nucleic acid sequence under a first set of conditions, and the The second region comprises a marker sequence that is located 5 'to the first region of the labeled oligonucleotide. The target hybridization sequence has a free 3 'hydroxyl group that can be enzymatically extended in the presence of a DNA polymerase in a template dependent manner. The labeled oligonucleotide has an active conformation that allows the target hybridizing sequence to hybridize to the target nucleic acid sequence and an inactive conformation that blocks the target hybridizing sequence from hybridizing to the target nucleic acid sequence. The inactive conformation is generally formed under less stringent conditions than the conditions for the formation of the active conformation of the labeled oligonucleotide.

The inactive conformation of the tagged oligonucleotide can be formed by hybridizing a tag closure sequence to the target hybridization sequence of the tagged oligonucleotide. The tag closure sequence may be a discrete molecule or may be linked to the tagged oligonucleotide via a spacer that links the 3 'end or 5' end of the tag closure sequence to the 5 'end of a region of the tagged oligonucleotide that contains a marker sequence (labeled primer oligonucleotide) or a promoter sequence located 5 'to a marker sequence (labeled promoter oligonucleotide), whereby a self-hybridizing hairpin tag molecule is formed comprising the tagged oligonucleotide. The spacer does not include nucleotide bases that can be copied by a polymerase, and is preferably a non-nucleotide spacer comprising non-nucleotide constituents. Suitable non-nucleotide spacers for joining the tag closure sequence to the tagged oligonucleotide include abasic nucleotides and polyethylene glycol. Other suitable spacers include nucleotide analogs, such as LNAs and 2'-O-Me. Association kinetics are best when the tag closure sequence and the target hybridization sequence of the tagged oligonucleotide are contained in the same molecule.

Under selective conditions, the tag closure sequence can hybridize to the target hybridization sequence of the tagged oligonucleotide in an antiparallel orientation, as shown in Figures 2, 4, 6, 8, 10, 11, 12, 15 and 16 , or in a parallel orientation, as shown in Figures 13 and 14. If the marker closure sequence is a discrete molecule, as illustrated in Figures 11 and 12, or is attached to the labeled oligonucleotide via a non-nucleotide spacer attached to its 5 'end, as illustrated in Figures 2, 4, 6, 8, 10, 15 and 16, the marker closure sequence is preferably modified to prevent primer extension by a DNA polymerase, such as by placing a blocking moiety at its 3 'end. Suitable blocking moieties are described herein. When hybridizing in an antiparallel orientation, as illustrated in Figures 13 and 14, the 3'-terminal base of the tag closure sequence preferably hybridizes to the 3'-terminal base of the target hybridization sequence of the labeled oligonucleotide. . More preferably, the marker closure sequence is modified to prevent primer extension by a DNA polymerase.

The length and base content of the tag closure sequence are selected so that hybridization of the tagged oligonucleotide to the target nucleic acid sequence is favored under a first set of conditions, and when the tagged oligonucleotide is not hybridized to the target nucleic acid sequence. target nucleic acid sequence, such that the marker closure sequence can form a stable hybrid with the target hybridizing sequence under a second set of less stringent conditions. The marker closure sequence should be selected so that it does not easily shift from the hybridization sequence to the target under the amplification conditions to which it may be subjected. In general, the marker closure sequence will hybridize to 5 to 20 contiguous or non-contiguous bases of the target hybridization sequence. Suitable marker closure sequences preferably range in length from 5 to 15 bases. To favor the sequence of hybridizing the target to the target nucleic acid in the first set of conditions, the marker closure sequence may include, for example, one or more abasic nucleotides, base mismatches, or non-base pair members. they are canonical. The marker closure sequences are preferably selected to specifically hybridize to the target hybridization sequence more strongly than any nonspecific interaction with other nucleic acids present in an amplification reaction.

Following inactivation, inactive labeled oligonucleotides are preferably removed from the sample to limit unintended interactions with target nucleic acid sequences that are introduced into the sample from a potentially contaminating source. Removal can be accomplished by immobilizing the target nucleic acids in a sample with a solid support, and

ES 2 358 296 T3 then removing other components from the sample, including inactivated labeled oligonucleotides. To ensure that inactive labeled oligonucleotides are removed, the number of nonspecific interactions between the solid support and nucleic acids present in the sample should be limited. Any known solid support can be used for sample processing, such as matrices and particles that are free in solution. Especially preferred supports are magnetic spheres that are monodisperse (ie 5% size uniformity), whereby consistent results are provided, which is especially advantageous for use in an automated procedure.

Especially preferred amplification techniques for incorporating the labeled oligonucleotides of the present invention include isothermal amplification reactions, such as TMA and variations of TMA, such as real-time TMA, which incorporate one or more features of the methods described by Becker et al. al., pub. Sun. by pat. from USA US 2006-0046265 A1, and Becker et al., sol. by pat. U.S. Serial No. 11 / 681.104. For example, certain preferred methods of real-time TMA include the use of blocking moieties, termination moieties, and / or other modifying moieties that provide improved sensitivity and accuracy to the TMA procedure.

The promoter oligonucleotides can be modified to prevent DNA synthesis from them. For example, a promoter oligonucleotide may comprise a blocking moiety attached to its 3 'end to prevent primer extension in the presence of a polymerase. In one example, at least about 80% of the oligonucleotides present in the amplification reaction that comprise a promoter further comprise a 3 'blocking moiety. In another embodiment, at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the oligonucleotides provided to the amplification reaction that comprise a promoter are further modified to comprise a blocking remainder in 3 '. In another embodiment, any oligonucleotide used in an amplification reaction of the present disclosure that comprises a promoter sequence further comprises a blocking moiety at the 3 'end.

Certain 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 indeterminate 3 'and 5' ends from the desired RNA target sequence. The target nucleic acid is treated with a primer oligonucleotide that has a base region complementary enough to a 3 'end of the target RNA sequence to hybridize to it and, as discussed above, further comprises a heterologous marker sequence in the first primer extension reaction. The primer oligonucleotides are designed to hybridize to a suitable region of any desired target sequence, according to primer design methods known to those of ordinary skill in the art. Although the presence of the marker sequence in a primer oligonucleotide can alter the binding characteristics of a target hybridization region to a target nucleic acid sequence, the skilled person in the molecular arts can easily design primer oligonucleotides that contain both regions hybridization to the target as marker sequences that can be used according to the methods described herein. Suitable priming oligonucleotides are described in more detail herein. In addition, the 5 'end of a primer oligonucleotide (preferably not a labeled primer oligonucleotide) may include one or more modifications that enhance the binding properties (eg, hybridization or base stacking) of the primer oligonucleotide to a DNA elongation product or an RNA amplification product, as discussed in more detail below, provided that the modifications do not substantially interfere with the priming function of the primer oligonucleotide or with the cleavage of an RNA amplification product to which the primer oligonucleotide is hybridized. The 3 'end of the primer oligonucleotide is extended by a suitable DNA polymerase, eg, an RNA-dependent DNA polymerase (reverse transcriptase) in an extension reaction by using the target RNA sequence or amplification product as template to provide a DNA primer extension product that is complementary to the RNA template or amplification product.

The DNA primer extension products are separated (at least partially) from an RNA template through the use of an enzyme that degrades the RNA template or amplification product. Suitable enzymes, i.e. selective RNases, are those that act on the RNA strand of an RNA: DNA complex, and include enzymes that comprise RNase H activity. Some reverse transcriptases include RNase H activity, including those derived from Moloney murine leukemia virus and avian myeloblastosis virus. According to preferred amplification embodiments, the selective RNase can be provided as an RNase H activity of a reverse transcriptase, or it can be provided as a separate enzyme, eg, as an E. coli RNase H or a T. thermophilus RNase H. Other enzymes that selectively degrade RNA present in a double-stranded aRN: DNA molecule can also be used.

When the target sequence is DNA, a DNA primer extension product can be separated from the template by treating the target nucleic acid with a displacer oligonucleotide. The displacer oligonucleotide has a priming function, and is designed to hybridize to nucleic acid

ES 2 358 296 T3 target in position 5 'of the primer oligonucleotide (referred to as the direct primer oligonucleotide in this embodiment). In position 5 'means that the 3' end of the displacer oligonucleotide hybridizes to the target nucleic acid 5 'to the 3' end of the forward primer oligonucleotide. Thus, the displacer oligonucleotide and the forward primer oligonucleotide can hybridize to overlapping or different regions of the target nucleic acid. In preferred embodiments, the 3 'end of the displacer oligonucleotide is adjacent or spaced up to 5 to 35 bases apart from the 5' end of the forward primer oligonucleotide relative to the target nucleic acid (i.e., the target nucleic acid has up to 5 to 35 contiguous nucleotides unbound located between the 3'-terminal base of the displacer oligonucleotide and the 5'-terminal base of the primer oligonucleotide when both oligonucleotides are hybridized to the target nucleic acid). The displacer oligonucleotide is generally 10 to 50 nucleotides in length and may include one or more 5 'end modifications that enhance the binding properties (eg, base stacking or hybridization) of the displacer oligonucleotide to the target nucleic acid, provided the modifications do not substantially interfere with the priming function of the displacer oligonucleotide. The displacer oligonucleotide and the forward primer oligonucleotide are designed to hybridize to the target nucleic acid under the same conditions. The target nucleic acid is preferably treated with the displacer oligonucleotide after the forward primer oligonucleotide has had sufficient time to hybridize to the target nucleic acid. Alternatively, the target nucleic acid is treated with the displacer oligonucleotide and the forward-priming oligonucleotide before exposing the mixture to a suitable polymerase to extend the 3 'end of the displacer oligonucleotide and the forward-priming oligonucleotide. In the presence of DNA polymerase, the 3 'end of the displacer oligonucleotide is extended in a template-dependent manner to form a second DNA primer extension product that displaces the first DNA primer extension product of the target nucleic acid, for which becomes available for hybridization to a promoter oligonucleotide. In an alternative approach, conditions could be established whereby the promoter oligonucleotide gains access to the first DNA primer extension product through invasion of the facilitated strand, for example, by local partial denaturation of DNA (e.g. g., AT-rich regions), low salinity conditions, and / or the use of DMSO and / or osmolytes, such as betaine. The promoter oligonucleotide of this embodiment is the same as described above and is similarly modified to prevent the promoter oligonucleotide from functioning as a primer oligonucleotide for a DNA polymerase (eg, the promoter oligonucleotide includes a blocking moiety at its 3 'end).

In certain embodiments, the methods of the present invention further comprise treating the target nucleic acid as described above to limit the length of the DNA primer extension product to a certain desired length. Such length limitation is generally accomplished through the use of a binding molecule that hybridizes or otherwise binds to the RNA target nucleic acid adjacent or near the 5 'end of the desired target sequence. In certain embodiments, a binding molecule comprises a region of bases. The base region can be DNA, RNA, a DNA: RNA chimeric molecule, or an analog thereof. Binding molecules that comprise 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 that comprises a terminator oligonucleotide or a terminator protein that binds to RNA and prevents extension of the primer beyond its binding region, or a binding molecule comprising a modifier molecule, for example, a modifier oligonucleotide such as a digest oligonucleotide that directs the hydrolysis of that part of the target RNA hybridized to the digest oligonucleotide, or a sequence specific nuclease that cuts the RNA target.

Illustrative termination oligonucleotides of the present disclosure have a 5 'base region sufficiently complementary to the target nucleic acid in a region adjacent, or close to, or overlapping the 5' end of the target sequence, as to hybridize to he. In certain embodiments, a terminator oligonucleotide is synthesized to include one or more modified nucleotides. For example, certain terminator oligonucleotides comprise one or more 2'O-ME ribonucleotides, or are completely synthesized with 2'-O-ME ribonucleotides. See, eg, Majlessi et al. (1998) Nucleic Acids Res., 26, 2224-2229. A terminator oligonucleotide also generally comprises a blocking moiety at its 3 'end to prevent the terminator oligonucleotide from functioning as a primer for a DNA polymerase. In certain embodiments, the 5 'end of a terminating oligonucleotide overlaps and is complementary to at least about 2 nucleotides from the 5' end of the target sequence. In general, the 5 'end of a terminator oligonucleotide of the present invention overlaps and is complementary to at least 3, 4, 5, 6, 7, or 8 nucleotides from the 5' end of the target sequence, but not more around 10 nucleotides from the 5 'end of the target sequence. (As used herein, the term "end" refers to a 5 'or 3' region of an oligonucleotide, nucleic acid, or nucleic acid region that includes, respectively, the 5'- or 3'-terminal base of the oligonucleotide, nucleic acid or nucleic acid region). Suitable terminator oligonucleotides are described in more detail herein.

A single-stranded DNA primer extension product, or first DNA primer extension product, having a defined 3 'end or an indeterminate 3' end, is treated

ES 2 358 296 T3 with a promoter oligonucleotide comprising a first region sufficiently complementary to a 3 'region of the DNA primer extension product to hybridize therewith, a second region comprising a promoter for an RNA polymerase, e.g. eg, T7 polymerase, which is located 5 'to the first region, eg, immediately 5' to or spaced from the first region, and modified to prevent the promoter oligonucleotide from functioning as a primer for a DNA polymerase (eg, the promoter oligonucleotide includes a blocking moiety attached to its 3 'end). After identifying a desired first hybridization region, one of ordinary skill in the art can construct suitable promoter oligonucleotides using only routine procedures. Those of ordinary skill in the art will readily understand that a promoter region has certain nucleotides that are necessary for recognition by a given RNA polymerase. Additionally, certain nucleotide variations in a promoter sequence could improve promoter performance with a given enzyme, including the use of insertion sequences.

Insertion sequences can be placed between the first and second promoter oligonucleotide regions and function to increase amplification rates. (The tag sequence of a tagged promoter oligonucleotide can provide this beneficial effect). The improved amplification rates can be attributed to several factors. First, because an insertion sequence increases the distance between the 3 'end and the promoter sequence of a promoter oligonucleotide, it is less likely that a polymerase, eg, reverse transcriptase, bound to the 3' end of the oligonucleotide promoter interferes with the binding of RNA polymerase to the promoter sequence, thereby increasing the rate at which transcription can be initiated. Second, the selected insert sequence can itself improve the speed of transcription by functioning as a better template for transcription than the target sequence. Third, because the RNA polymerase will initiate transcription at the insertion sequence, the primer extension product synthesized by the primer oligonucleotide, using the RNA transcription product as a template, will contain the complement of the sequence. insert near the 3 'end of the primer extension product. By providing a larger target binding region, i.e. one that includes the complement of the insertion sequence, the promoter oligonucleotide can bind to the primer extension product faster, thereby leading to the production of transcription products. of additional RNAs in less time. The insertion sequences are preferably 5 to 20 nucleotides in length, and should be designed to minimize intramolecular folding and intermolecular binding with other oligonucleotides present in the amplification reaction mixture. Programs that help minimize secondary structure are well known in the art, and include Michael Zucker's mfold computer support for predicting RNA and DNA secondary structure using nearest neighbor thermodynamic rules. The latest version of Michael Zucker's mfold computer support can be obtained from the Internet page www.bioinfo.rpi.edu/applications/mfold by using a hypertext transfer protocol (http) in the URL. Useful insertion sequences can be identified using in vitro selection methods well known in the art without engaging in more than routine experimentation.

The promoter oligonucleotide assay with variations of the promoter sequences is easily carried out by the skilled artisan using routine methods. Furthermore, if it is desired to use a different RNA polymerase, the promoter sequence in the promoter oligonucleotide is easily replaced by a different promoter. Substitution of the different promoter sequences is within the knowledge and ability of those of ordinary skill in the art. For real-time TMA, the promoter oligonucleotides provided to the amplification reaction mixture are modified to prevent efficient initiation of DNA synthesis from their 3 'ends, and preferably comprise a blocking moiety attached to their 3' ends. . Furthermore, the terminator oligonucleotides and the protective oligonucleotides, and even the probes used in certain embodiments also optionally comprise a blocking moiety attached to their 3 'ends.

When a terminator oligonucleotide is used, the first region of the promoter oligonucleotide is designed to hybridize to a desired 3 'end of the DNA primer extension product with substantial precision, but not necessarily exact. Subsequently, the second region of the promoter oligonucleotide can act as a template, allowing the first DNA primer extension product to be further extended to add a base region complementary to the second region of the promoter oligonucleotide, that is, the region that it comprises the promoter sequence, which makes the promoter double-stranded. An RNA polymerase that recognizes the promoter binds to the promoter sequence, and initiates transcription of multiple RNA copies complementary to the DNA primer extension product, the copies of which are substantially identical to the target sequence. Substantially identical means that the multiple RNA copies may have additional nucleotides 5 'or 3' of the target sequence, or they may have fewer nucleotides 5 'or 3' of the target sequence, depending on, eg, the boundaries of the target sequence, the transcription initiation point, or whether the primer oligonucleotide comprises additional nucleotides 5 'of the primer region (e.g., a bonded shield as described herein). When the target sequence is DNA, the sequence of the RNA copies is described herein as substantially identical to the target sequence. Must be

ES 2 358 296 T3 understand, however, that an RNA sequence that has uridine residues in place of the thymidine residues of the target DNA sequence still has a substantially identical sequence. The RNA transcripts thus produced can be automatically recirculated in the above system without further manipulation. Thus, this reaction is autocatalytic. In those embodiments where no binding molecule or other means is used to terminate the primer extension reaction, the first region of the promoter oligonucleotide is designed to hybridize to a selected region of the first DNA primer extension product that expected to be 5 'to the 3' end of the first DNA primer extension product, but because the 3 'end of the first DNA primer extension product is indeterminate, the region to which the promoter oligonucleotide hybridizes will likely not be the actual 3' end of the first DNA primer extension product. According to this embodiment, it generally occurs that at least the 3'-terminal 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 likely will not be further extended to form a double-stranded promoter.

The formation of a double-stranded promoter sequence by means of the extension of a template nucleic acid is not necessary to allow the initiation of transcription of RNA complementary to the first DNA primer extension product. The resulting first RNA products are substantially identical to the target sequence, which has a 5 'end defined by the transcription initiation point, and a 3' end defined by the 5 'end of the first DNA primer extension product. . A sufficient number of RNA first products are produced to be automatically recirculated in the system without additional manipulation. The primer oligonucleotide hybridizes to the 3 'end of the first RNA products, and is extended by a DNA polymerase to form a second DNA primer extension product. Unlike the first DNA primer extension product formed without the use of a terminator oligonucleotide or other binding molecule, the second DNA primer extension product has a defined 3 'end that is complementary to the 5' ends of the first RNA products. The second DNA primer extension product is separated (at least partially) from the RNA template through the use of an enzyme that selectively degrades the RNA template. The second single-stranded DNA primer extension product is then treated with a promoter oligonucleotide as described above, and the second region of the promoter oligonucleotide acts as a template, allowing the second DNA primer extension product to be extended further. to add a base region complementary to the second region of the promoter oligonucleotide, i.e., the region comprising the promoter sequence, which makes the promoter double-stranded. An RNA polymerase that recognizes the promoter binds to the promoter sequence, and initiates transcription of multiple second RNA products complementary to the second DNA primer extension product, and substantially identical to the target sequence. The second RNA transcripts thus produced are automatically recirculated in the above system without further manipulation. Thus, this reaction is autocatalytic.

In any of the embodiments described above, once a desired region for the target sequence is identified, that region can be analyzed to determine where degradation by selective RNase will optimally cause cutting or removal of RNA sections from the RNA double-stranded molecule. : DNA. Analysis can be carried out to determine the effect of RNase degradation of the target sequence by the RNase H activity present in AMV reverse transcriptase or MMLV reverse transcriptase, by a selective enzyme added exogenously with an RNase activity, eg, E. coli RNase H, or selective enzymes with RNase activity from other sources, and by combinations thereof. Following such analyzes, the primer oligonucleotide can be selected to hybridize to a section of RNA that is not substantially degraded by the selective RNase present in the reaction mixture, because substantial degradation at the binding site for the primer oligonucleotide it could inhibit the initiation of DNA synthesis and prevent optimal primer extension. In other words, a primer oligonucleotide is generally selected to hybridize to a region of the RNA target nucleic acid or the complement of the DNA target nucleic acid, located so that when the RNA is subjected to selective RNase degradation, there is no substantial degradation that could prevent formation of the primer extension product.

Conversely, the promoter oligonucleotide hybridization site can be chosen such that sufficient degradation of the DNA strand occurs to allow efficient hybridization of the promoter oligonucleotide to the DNA strand. Generally, only portions of RNA are removed from the RNA: DNA double-stranded molecule by selective RNase degradation, and thus some parts of the RNA strand will remain on the double-stranded molecule. Selective RNase degradation in the RNA strand of an RNA: DNA hybrid results in the dissociation of small pieces of RNA from the hybrid. The positions at which RNA is selectively degraded can be determined by standard hybridization analysis. Thus, a promoter oligonucleotide can be selected that will bind more efficiently to DNA after degradation by

ES 2 358 296 T3

RNase selective, that is, it will bind to areas where RNA fragments are selectively removed.

Promoters or promoter sequences suitable for incorporation into the promoter oligonucleotides used in the methods of the present invention are nucleic acid sequences (natural, synthetically produced, or a product of a restriction digest) that are specifically recognized by an RNA. polymerase that recognizes and binds to that sequence and initiates the transcription process, by which RNA transcripts are produced. Typical, known, and useful promoters include those that are recognized by certain bacteriophage polymerases, such as those from bacteriophage T3, T7, and SP6, and a promoter from E. coli. The sequence can optionally include nucleotide bases that extend beyond the particular RNA polymerase recognition site that can confer added 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 initiation sequence are especially suitable for use.

Suitable DNA polymerases for use according to the methods of the disclosure include reverse transcriptases. Especially suitable 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 RNase activity in the amplification reaction is provided by reverse transcriptase - no additional selective RNase is added. However, in certain situations it may also be useful to add a selective exogenous RNase, such as E. coli RNase H. Although the addition of an exogenous selective RNase is not necessary, under certain conditions the RNase H activity present, eg, in AMV reverse transcriptase can be inhibited or inactivated by other components present in the reaction mixture. In such situations, the addition of an exogenous selective RNase may be desirable. For example, when relatively large amounts of heterologous DNA are present in the reaction mixture, the native RNase H activity of the AMV reverse transcriptase can be inhibited to some extent, thereby reducing the number of copies of the target sequence produced. In situations where the target nucleic acid comprises only a small portion of the nucleic acid present (eg, when the sample contains significant amounts of heterologous DNA and / or RNA), it is especially useful to add a selective exogenous RNase. See, eg, Kacian et al, pat. U.S. No. 5,399,491.

The RNA amplification products produced by the methods described above can serve as templates to produce additional amplification products related to the target sequence by means of the mechanisms described above. The system is autocatalytic, and amplification occurs without the need to repeatedly modify or change reaction conditions, such as temperature, pH, ionic strength, and the like. These methods do not require an expensive thermal cycler apparatus, nor do they require multiple additions of enzymes or other reagents during the course of an amplification reaction.

As noted above, the methods of the present disclosure are useful in assays to detect and / or quantify specific target nucleic acid sequences in clinical, water, environmental, industrial, beverage, food, seed stock, and other samples. , or to produce a large number of RNA amplification products from a specific target sequence for a variety of uses. For example, the present invention is useful for screening clinical samples (eg, blood, urine, feces, saliva, semen, or cerebrospinal fluid), food, water, laboratory and / or industrial samples, for the presence of specific nucleic acids, specific organisms (eg, through the use of species-specific oligonucleotides), and / or specific classes of organisms in applications such as sterility testing (eg, through the use of universal oligonucleotides). The present description can be used to detect the presence, for example, of viruses, bacteria, fungi, or parasites.

The amplification product can be detected by any conventional means. For example, the amplification product can be detected by hybridization with a detectably labeled probe and by measuring the resulting hybrids. Design criteria when selecting probes for the detection of particular 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, pat. U.S. No. 6,150,517. Hogan teaches that probes should be designed to maximize homology to the target sequence (s) and to minimize homology to potential non-target sequences. To minimize stability with non-target sequences, Hogan notes that guanine and cytosine rich regions should be avoided, that the probe should span as many destabilizing mismatches as possible, and that the length of perfect complementarity to a sequence should be minimized. non objective. Rather, the stability of the probe with the target sequence (s) should be maximized, adenine and thymine rich regions should be avoided, probe: target hybrids are preferably terminated with

ES 2 358 296 T3 guanine and cytosine base pairs, extensive self-complementarity should be avoided in general, and the melting temperature of the probe: target hybrids should be around 2-10 ° C higher than the assay temperature .

In a particular embodiment, the amplification product can be tested by the protection by hybridization assay (HPA), which involves hybridizing a chemiluminescent oligonucleotide probe to the target sequence, eg, an acridinium ester (AE) -labeled probe. , selectively hydrolyze the chemiluminescent marker present on the unhybridized probe, and measure the chemiluminescence produced from the remaining probe in a luminometer. See, eg, Arnold et al., Homogenous Protection Assay, pat. No. 5,283,174 and NORMAN C. NELSON ET AL., NONISOTOPIC PROBING, BLOTTING, AND SEQUENCING, ch. 17 (Larry J. Kricka ed., 2^to ed. nineteen ninety five).

In additional embodiments, the present description provides a quantitative study of the real-time amplification process by the methods described herein. Studying a real-time amplification process involves determining the amount of amplicon in the reaction mix continuously or periodically during the amplification reaction, and the determined values ​​are used to calculate the amount of target sequence initially present in the sample. . There are a variety of methods for determining the amount of target sequence present in a sample based on real-time amplification. These include those described by Wittwer et al., Method for Quantification of an Analyte, pat. US Patent No. 6,303,305, and Yokoyama et al., Method for Assaying Nucleic Acid, pat. U.S. No. 6,541,205. Another method for determining the amount of target sequence initially present in a sample, but not based on real-time amplification, is described in Ryder et al., Method for Determining Pre-Amplification Levels of a Nucleic Acid Target Sequence from Post -Amplification Levels of Product, pat. U.S. No. 5,710,029. Amplification products can be detected in real time through the use of various self-hybridizing probes, most of which have a stem-loop structure. Such self-hybridizing probes are labeled so that they emit detectable signals differentially, depending on whether the probes are in a self-hybridizing state or in an altered state through hybridization to a target sequence. By way of example, molecular torches are a type of self-hybridizing probe that include different regions of self-complementarity (called target binding domain and target closure domain) that are connected by a binding region (e.g. ., a non-nucleotide spacer) and that hybridize to each other under predetermined hybridization assay conditions. In a preferred embodiment, molecular torches contain single-stranded base regions in the target binding domain that are 1 to about 20 bases in length and are accessible for hybridization to a target sequence present in an amplification product under conditions of chain offset. Under strand displacement conditions, hybridization of the two complementary regions (which may be completely or partially complementary) of the molecular torch is favored, except for the presence of the target sequence, which will bind to the single stranded region present in the target binding domain and will displace all or part of the target's closure domain. The target binding domain and target closure domain of a molecular torch include a detectable marker or a pair of interacting markers (eg, luminescent / quenching agent) positioned so that a different signal is produced when the torch molecular is self-hybridized and when the molecular torch hybridizes to the target sequence, thus, the detection of probe: target double-stranded molecules is allowed in a test sample in the presence of unhybridized molecular torches. Molecular torches and a variety of types of interacting marker pairs are described in Becker et al., Molecular Torches, pat. from USA No. 6,534,274.

Another example of a detection probe that has self-complementarity is a molecular beacon. Molecular beacons include nucleic acid molecules that have a sequence complementary to the target, an affinity pair (or nucleic acid arms) that maintain the probe in a closed conformation in the absence of a target sequence present in an amplification product, and a marker pair that interacts when the probe is in a closed conformation. Hybridization of the target sequence and the sequence complementary to the target separates the members of the affinity pair, thereby shifting the probe to an open conformation. The shift to the open conformation is detectable due to the reduced interaction of the marker pair, which can be, for example, a fluorophore and a quencher (eg, DABCYL and EDANS). Molecular beacons are described in Tyagi et al., Detectably Labeled Dual Confirmation Oligonucleotide Probes, Assays and Kits, US Patent No. 5,925,517, and Tyagi et al., Nucleic Acid Detection Probes Having Non-FRET Fluorescence Quenching and Kits and Assays Including Such Probes, US Patent No. 6,150,097.

Other self-hybridizing probes are known to those of ordinary skill in the art. By way of example, one could adapt probe binding pairs having interacting markers, such as those described in Morrison, Competitive Homogenous Assay, US Patent No. 5,928,862 and Gelfand et al., Pat. US No. 5,804,375 for PCR reactions, for use herein. Additional detection systems include molecular switches, such as those described by Arnold et al., Oligonucleotides Comprising a Molecular Switch, pub. Sun. by pat. US No. US 2005-0042638 A1. And other probes, such as those comprising intercalating dyes

ES 2 358 296 T3 and / or fluorochromes, could be useful for the detection of amplification products in the present disclosure. See, eg, Ishiguro et al., Method of Detecting Specific Nucleic Acid Sequences, US Patent No. 5,814,447.

In methods where the initial target sequence and the RNA transcript share the same sense, it may be desirable to initiate amplification before adding the probe for real-time detection. Adding the probe before starting an amplification reaction can slow down the rate of amplification, as the probe that binds to the initial target sequence has to be displaced or otherwise removed during the primer extension step to complete a product. of primer extension having the complement of the target sequence. The initiation of amplification is determined by the addition of amplification enzymes (eg, a reverse transcriptase and an RNA polymerase).

In addition to the methods described herein, the present disclosure is directed to kits comprising one or more of the reagents necessary to carry out the methods of the present disclosure. Kits comprising various components used to carry out the present disclosure can be configured for use in any procedure that requires amplification of target nucleic acid molecules, and such kits can be adapted for various end users. Suitable kits can be prepared, for example, for microbiological analysis, blood screening, disease diagnosis, water analysis, product marketing or sterility analysis, environmental or industrial analysis, analysis of food or drink, or for general laboratory use. The kits provide one or more of the components necessary to carry out the nucleic acid amplifications according to the invention. Kits can include reagents suitable for amplifying nucleic acids from a particular target, or they can include reagents suitable for amplifying multiple targets. Kits can further provide reagents for the real-time detection of one or more nucleic acid targets in a single sample, eg, one or more self-hybridizing probes as described above. The kits may comprise a container that may be compartmentalized to receive in a confined space one or more containers such as vials, test tubes, wells, and the like. Preferably, at least one such container contains one or more components or a mixture of components necessary to carry out the amplification methods of the present invention.

A kit according to an embodiment of the present disclosure may include, for example, in one or more containers, a labeled oligonucleotide, alone or in combination with a label closure oligonucleotide or linked to a label closure sequence, a binding molecule or other means of terminating a primer extension reaction, and, optionally, an extension oligonucleotide and / or a protective oligonucleotide. If real-time detection is used, the container (s) may include one or more reagents for the real-time detection of at least one target nucleic acid sequence in a single sample, for example, one or more self-hybridizing probes such as those described above. Another container may contain an enzymatic reagent, such as a thermostable DNA polymerase to carry out a PCR or RT-PCR reaction, or a mixture of a reverse transcriptase (with or without RNase H activity), an RNA polymerase, and optionally an additional selective RNase enzyme for a transcription-based amplification reaction. These enzymes can be provided in concentrated form or at working concentration, usually in a form that promotes enzyme stability. The enzyme reagent can also be provided in lyophilized form. See Shen et al., Stabilized Enzyme Compositions for Nucleic Acid Amplification, US Patent No. 5,834,254. One or more other containers may contain an amplification reagent in concentrated form, eg, 10X, 50X, or 100X, or at the working concentration. An amplification reagent will contain one or more of the components necessary to carry out the amplification reaction, eg, a buffer, MgCl2, KCl, dNTPs, rNTPs, EDTA, stabilizing agents, etc. Certain components, eg, MgCl2 and rNTPs, can be provided separately from the remaining components, allowing the end user to titrate these reagents for more optimized amplification reactions. One or more other containers may be included for the detection of the amplification products, including one or more labeled oligonucleotide probes. The probes can be labeled in a number of alternative ways, eg, with radioactive isotopes, fluorescent labels, chemiluminescent labels, nuclear labels, bioluminescent labels, intercalating dyes, or enzyme labels. In certain embodiments, a kit will also include one or more containers containing one or more positive and negative control target nucleic acids, which can be used in amplification experiments to validate the amplifications of assays performed by the end user. In certain cases, one or more of the reagents listed above can be combined with an internal control. Of course, it is also possible to combine one or more of these reagents in a single tube or other containers. Suitable holders for use with the description, eg, test tubes, multi-tube units, multi-well plates, etc., can also be supplied with the kits. Finally, a kit of the present invention may include one or more instruction manuals.

Kits can contain virtually any combination of the components set forth above or described elsewhere herein. As one skilled in the art would recognize, the components supplied with the kits of the invention will vary with the use to which

ES 2 358 296 T3 are intended for the equipment, and the end user. Thus, equipment can be specifically designed to perform various functions set forth in this application, and the components of such equipment will vary accordingly.

The present disclosure is further directed to various oligonucleotides including, for example, the target specific oligonucleotides exemplified below. It should be understood that the oligonucleotides of the present disclosure can be DNA, RNA, DNA: RNA chimeric molecules and the analogs thereof, and in any case, the RNA equivalents of the DNA oligonucleotides and the DNA equivalents are included. of RNA oligonucleotides.

Detection probes can include, for example, an acridinium ester marker, or labeled, self-hybridizing regions that flank the sequence that hybridizes to the target sequence. In various embodiments, these labeled oligonucleotide probes are optionally or preferably synthesized to include at least one modified nucleotide, eg, a 2'-O-ME ribonucleotide; or these labeled oligonucleotide probes are optionally or preferably completely synthesized from modified nucleotides, eg, 2'-O-ME ribonucleotides.

EXAMPLES

Examples illustrating certain aspects and embodiments of the invention are provided below.

Unless otherwise indicated, the oligonucleotides and modified oligonucleotides of the following examples were synthesized using standard phosphoramidite chemistry, various methods of which are well known in the art. See, eg, Carruthers et al. (1987) Meth. Enzymol. 154, 287. Unless otherwise indicated herein, the modified nucleotides were 2'-O-ME ribonucleotides, which were used in the synthesis as their phosphoramidite analogs.

EXAMPLE 1

SELECTIVE AMPLIFICATION OF HCV USING LABELED OLIGONUCLEOTIDES IN A REAL-TIME TMA REACTION

The following series of experiments were carried out to determine whether the use of a labeled oligonucleotide to modify a target nucleic acid sequence in a nucleic acid sample of interest prior to a transcription-mediated amplification reaction would allow selective amplification of the target nucleic acid sequence contributed by the nucleic acid sample of interest, while the target nucleic acid sequence contributed by sources other than the nucleic acid sample of interest would not be amplified.

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

I. OLIGONUCLEOTIDES

Unless otherwise indicated, oligonucleotides were synthesized using an Expedite ™ 8909 DNA synthesizer (PerSeptive Biosystems, Framingham, MA) using standard phosphoramidite chemistry. See, eg, Carruthers et al. (1987) Meth. Enzymol. 154, 287. Sequences are 5'-to-3 '. The blocking moiety, if present, is at the 3 'end.

1. Labeled priming oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACAGGCATTGAGCGGGTTGATCCAAGAAAGGAC (SEQ ID NO: 1); 12 pmol / reaction

2. Priming Oligonucleotide:

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

3. Promoter Oligonucleotide:

ATTTAATACGACTCACTATAGGGAGACCACAACGGTTTCTAGCCATGGCG

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

Blocking Moiety: A 3'-to-3 'linkage prepared using 3'-dimethyltrityl-N-benzoyl-2'deoxycytidine, 5'-succinoyl-long chain alkylamino-CPG (Glen Research Corporation, Sterling, VA; cat. no.20-0102-01)

ES 2 358 296 T3

Four. Termination Oligonucleotide:

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

Blocking Rest: The same as the promoter oligonucleotide

5. Prolongator Oligonucleotide:

TGTCGTGCAGCCTCCAGGACCCCCCCTCCCG GGAGAGCCATA (SEQ ID NO: 5); 12 pmol / reaction

Blocking Rest: The same as the promoter oligonucleotide

6. First Capture Probe:

GmGmGmCmAmCmUmCmGmCmAmAmGmCmAmmCmCmCmUmTTTAAAAAAAAAAAAAAA AAAAAAAAAAAAAAA (SEQ ID NO: 6); 3 pmol / reaction

7. Second Capture Probe:

CmAmUmGmGmUmGmCmAmCmGmGmUmCmUmAmCmGmTTTAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA (SEQ ID NO: 7); 3 pmol / reaction

8. Detection Probe:

CmGmUmUmCmCmGmCmAmGmAmCmCmAmCmUmAmUm (Spacer) GmAmAmCmGm (SEQ ID NO: 8); 4 pmol / reaction

Probe Type: Molecular Torch

Spacer: 9-O-Dimethoxytrityl-triethylene glycol, 1 - [(2-cyanoethyl) - (N, N-diisopropyl)] - phosphoramidite (Glen Research Corporation, Sterling, VA; Cat. # 10-1909-90)

5 'Marker: 6-Carboxyfluorescein (FAM) (BioGenex, San Ramon, CA; Cat. # BGX-300801)

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

II. REAGENTS AND OTHER PROTOCOL INFORMATION

1. Amplification Reagent. Amplification Reagent or AMP Reagent consisted of 11.6 mM Trizma® base buffer, 15 mM Trizma® hydrochloride buffer, 25 mM MgCl2, 23.3 mM KCl2, 3.33% (v / v) glycerol, acetate 0.05 mM zinc, 0.76 mM dATP, 0.76 mM dCTP, 0.76 mM dGTP, 0.76 mM dTTP, 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 at 8.0 at 22 ° C.

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

3. Wash Solution. The Wash Solution consisted of 10 mM HEPES, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v / v) ethanol, 0.02% (w / v) methyl paraben, 0.01% ( w / v) of propyl paraben, 150 mM NaCl, and 0.1% (w / v) of sodium dodecyl sulfate, pH 7.5.

Four. Conveyance. The Transport Medium consisted of 150 mM HEPES, 8% (w / v) lithium lauryl sulfate, and 100 mM ammonium sulfate, pH 7.5.

5. Target Capture Reagent. The Target Capture Reagent or TCR consisted of the components listed below. Additional information on the formulation of this mixture is described below in the Target Capture Reagent (IIIA) Procedure. The concentrations listed represent the final concentrations of the components after they have been combined with the magnetic particle solution. The magnetic particles were carboxylate modified Sera-Mag ™ MG-CM super-paramagnetic particles (Seradyn, Inc.,

ES 2 358 296 T3

Indianapolis, IN; cat no. 24152105-050250), 1 micron, covalently linked to 5 'amino modified oligo (dT) 14. The HEPES components, lithium hydroxide, lithium chloride, EDTA, lithium lauryl sulfate, and ammonium sulfate were introduced with the TCR solvent and transport medium.

First Capture Probe; 15.0 nM

Second Capture Probe; 15.0 nM

Marked Priming Oligonucleotide; 60.0 nM

Termination 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 sulphate; 37.5 mM

Seradyn Poly dT14 Magnetic Particles; 0.075 ug / uL

6. Transcription Buffer. The Transcription Buffer consisted of 0.2% lithium lauryl sulfate.

7. Transcript used. HCV transcript.

8. Product Numbers of Certain Materials or Equipment Used.

KingFisher ™ Plate (Thermo Labsystems, Franklin, MA; Cat. # 97002540)

MJ Research Microtiter Plate (Bio-Rad Laboratories, Inc., Hercules, CA; Cat. # HSP9665)

Solo HT Incubator (Thermo Labsystems, Franklin, MA; Cat. # 5161580)

KingFisher ™ Comb (Thermo Labsystems, Franklin, MA; Cat. # 97002510)

Eppendorf® Thermomixer R (Eppendorf North America; Westbury, NY; Cat. # 022670107 or 022670158)

DNA Engine Opticon® 2 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA; Cat. # CFB-3220)

9. Additional Information of the Protocol.

For the described experiments, 3.3 pL of transcription buffer containing the target was added to each 2.0 ml microtube in step B6 below. The labeled primer oligonucleotide and terminator oligonucleotide were in water prior to adding them to the 2.0 mL microtubes. The samples were vortexed for about 5 seconds. Incubation for 10 minutes at 60 ° C was generally considered sufficient to capture the transcript. The plates were kept at room temperature for 5 minutes after the 10 minute incubation to allow the plates to cool before the target capture steps. It was at this point also that the plates were transferred from the Solo HT incubator to the KingFisher system. The speed of the thermomixer was 1400 rpm.

III. TARGET CAPTURE PROTOCOL

A. Procedure for Target Capture Reagent (TCR).

The magnetic spheres were slowly mixed at room temperature (RT) for 45 minutes, and 150 pL of magnetic spheres were added to 5mL of TCR solvent (15 pg of spheres / reaction when 50 pL per sample was used). The solution was slowly mixed at room temperature for 35 minutes, at which time the capture probe was added to 5 mL of the TCR diluent (to a final concentration of 0.12 pmol / pL (6-pmol / 50 pL reaction).

ES 2 358 296 T3

B. Sample Preparation.

AMP Reagent was prepared containing the promoter oligonucleotide, the extender oligonucleotide, and the primer oligonucleotide (volume = 1600 pL). The solution was vortexed and placed at 2-8 ° C until needed. The detection probe was prepared in Enzyme Reagent and placed at 2-8 ° C until needed. Target dilutions were made in 0.2% LLS. 50 pL of TCR was transferred to 200 pL wells of a microplate. Each level of copies of the target, labeled primer oligonucleotide, and terminator oligonucleotide were added to 1.2 mL of 50% Transport Medium, 50% H2O in 2.0 mL microtubes. Target samples were vortexed and 150 pL transferred to a 200 pL well of a microplate (Plate 1) containing 50 pL TCR (each well contained zero or 1 million copies of HCV transcript plus appropriate amounts of labeled primer oligonucleotides and terminator oligonucleotides).

C. Target Capture Protocol.

The 200 pL microplate (Plate 1) was incubated at 60 ° C for 10 minutes using the Labsystems Solo HT Incubator (Plate 1), and the microplate was then placed at RT for 5 minutes (Plate 1). 200 pL microplates (Plates 2 and 3) were prepared with 200 uL Wash Reagent. An amplification plate (4-MJ research 96-well microtiter plate) was prepared with 30 pL of AMP Reagent per well. The 96-well comb was placed on Plate 1. All four plates were loaded onto the KingFisher 96 unit and the target capture protocol was initiated as follows.

Plate 1 was mixed for 5 minutes at a very slow speed, and the spheres were collected for 12 counts and then released onto Plate 2 for 10 seconds using a slow speed. Plate 1 was then mixed for 1 second using a very slow speed, the spheres were collected for 12 counts, and the spheres were released on Plate 2 for 10 seconds using a slow speed.

Plate 2 was mixed for 30 seconds at medium speed, and the spheres were collected for 12 counts and then released onto Plate 3 for 10 seconds using a very slow speed. Plate 2 was then mixed for 1 second at a very slow speed, and the spheres were collected for 12 counts and released onto Plate 3 for 10 seconds using a very slow speed.

Plate 3 was mixed for 30 seconds at medium speed, the spheres were collected for 12 counts, and the spheres were released on Plate 4 for 10 seconds using a medium speed. Plate 3 was then mixed for 1 second at a very slow speed, the spheres were collected for 12 counts and released onto plate 4 for 10 seconds using a medium speed.

The 96-well microtiter plate (Plate 4) was removed and transferred to the table, covered with a closure plate, and placed in the DNA Engine Opticon® 2 Real-Time PCR Detection System (Bio-Rad Laboratories; Hercules, CA) (real-time instrument).

D. Real Time TMA.

The real-time TMA was carried out as follows. The plate was incubated for 5 minutes at 42 ° C and then removed and placed in a thermomixer at 42 ° C. Each reaction well received a 10 pL aliquot of Enzyme Reagent. The microtiter plate was covered with an adhesive tape closure, gently shaken for 30 seconds in the thermomixer, and then placed in the real-time instrument at 42 ° C, where real-time assay monitoring began. The TTtime values, which served as indicators of the amount of amplicon synthesized, were determined from the monitored fluorescence signals. See Light et al., Pub. Sun. by pat. US 2006-0276972, paragraphs 506-549.

IV. RESULTS AND CONCLUSION

Experiments were carried out according to the procedures described above for the detection of an HCV transcript (8 replicates). The TCR in each assay contained the same labeled primer oligonucleotide. A target capture step was performed to bind the HCV transcript and remove the unhybridized labeled primer oligonucleotide and the unhybridized terminator oligonucleotide. After the target capture step, an AMP Reagent was contacted with the TCR spheres, and the AMP Reagent contained a primer oligonucleotide specific for the complement of the marker sequence. No labeled primer oligonucleotide was included in this step.

Eight replicates were tested for each condition. The detection probe was added via Enzyme Reagent at 4 pmol per reaction. The HCV AMP Reagent contained 12 pmol of

ES 2 358 296 T3 promoter oligonucleotide, 12 pmol of extender oligonucleotide and 12 pmol of priming oligonucleotide per reaction.

The first set of experiments compared the results of reactions in which no copies of the HCV transcript were added in the TCR or AMP Reagent with the results of reactions in which 1 x 10 were added.⁶ copies of the HCV transcript in the TCR. Figure 17 shows the crude curves for the HCV amplifications, in which no amount of target was added in the AMP Reagent. There was no detectable amplification when the HCV transcript was not added in the TCR or AMP Reagent, while the TTime for reactions containing 1 x 10⁶ copies of the HCV transcript in the TCR was 6.3 minutes. The values ​​of Ttime refer to the time of appearance (time in which a signal emerges above the background), and a summary of these values ​​for the experiments carried out is set forth in Table 1 below.

A second group of experiments compared the results of reactions in which 1 x 10⁶ copies of the HCV transcript in the AMP Reagent only with reactions in which 1 x 10⁶ copies of the HCV transcript in the TCR only. Figure 18 shows the crude curves for the HCV amplifications, in which the target was added in the AMP Reagent. There was no detectable amplification when the HCV transcript was added into the AMP Reagent, while the mean time for reactions containing 1 x 10⁶ copies of the HCV transcript in the TCR was 6.3 minutes (Table 1). The zero target in the tC samples was not amplified, even with 1 million copies of the HCV transcript added in the AMP Reagent.

A third group of experiments compared the results of reactions in which 1 x 10⁶ copies of the HCV transcript and the priming oligonucleotide labeled in the AMP Reagent (no copies of the HCV transcript in the TCR) with the results of the reactions in which 1 x 10 were given⁶ copies of the HCV transcript in the TCR and the primer oligonucleotide labeled in the AMP Reagent. Figure 19 shows that the mean Ttime for 1 million copies of HCV transcript present only in the target capture step with labeled primer oligonucleotides and terminator oligonucleotides added in the AMP Reagent was 7.2 minutes. Zero samples with target, terminator oligonucleotide, and labeled primer oligonucleotide added in the AMP Reagent also produced solid amplification with a mean time = 8.6 minutes (Table 1).

Table 1. Summary of TTime (TTimes and TTimes SD)

<td>Sample ID</td><td>Name of Target</td><td>Objective Qty</td><td>Total</td><td>Tz I heard</td><td>Tz 1-</td><td>TTaverage time</td><td>Time FROM</td>

<td>1 million target on 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, labeled non-T7 primer and terminator oligonucleotide in amp-x6.0</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 on 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>Zero target at TC, 1 million target at amp-x0.0</td><td>HCV</td><td> 0,00</td><td> 8</td><td> 8</td><td> 0</td><td>N / A</td><td>N / A</td>

<td>Zero target on TC, 1 million target, labeled non-T7 primer, and terminator oligonucleotide at amp-x0.0</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>Target Zero at TC-x0.0</td><td>HCV</td><td> 0,00</td><td> 8</td><td> 8</td><td> 0</td><td>N / A</td><td>N / A</td>

The results of these experiments demonstrate that only when the labeled primer oligonucleotide was present in the AMP Reagent along with the primer oligonucleotide did the zero TCR samples amplify when 1 million copies of HCV transcript were added to the AMP Reagent. Thus, the HCV transcript that enters the system through the AMP Reagent is not amplified unless the labeled primer oligonucleotide is also provided with the AMP Reagent.

ES 2 358 296 T3

The preceding Example demonstrated how a labeled primer oligonucleotide that hybridized to an HCV template could be used to selectively detect HCV nucleic acids in a sample of interest without interference from contaminating nucleic acid subsequently introduced in a capture step of the target. The following Example illustrates how a similar approach was used to detect bacterial nucleic acids in a sample of interest despite the presence of contaminating templates in the reagents used to carry out the amplification reaction. Advantageously, the uncomplexed labeled primer oligonucleotide was substantially non-existent in the reaction mixture at the time that the complex comprising the labeled primer oligonucleotide and the template came into contact with the DNA polymerase used in the amplification reaction. .

Example 2 below describes two procedures for amplifying E. coli rRNA nucleic acids, in which the procedures differed by the use of a labeled primer oligonucleotide and target capture. The first procedure employed an E. coli-specific unlabeled primer oligonucleotide in combination with a terminator oligonucleotide, a promoter oligonucleotide, and a detection probe. The second procedure employed a labeled primer oligonucleotide that had a complementary target sequence identical to that contained in the E. coli-specific unlabeled primer oligonucleotide from the first procedure, a marker specific primer oligonucleotide, as well as a terminator oligonucleotide. , a promoter oligonucleotide and a detection probe. The marker-specific primer oligonucleotide, which had a nucleotide sequence corresponding to a segment of HIV-1, hybridized to the complement of the marker sequence contained in the labeled primer oligonucleotide, but did not hybridize to the E rRNA template nucleic acid. coli or its complement. In the case of the second procedure, the terminator oligonucleotide, the promoter oligonucleotide, and the detection probe were identical to those used in the first procedure. As demonstrated below, amplification reactions that omitted the olig₆Labeled primer onucleotide were unable to distinguish between samples containing 0 and 10⁶ copies of a synthetic E. coli rRNA target. Conversely, the approach that included the use of a tagged priming oligonucleotide and target capture clearly distinguished samples containing 0 and 10³ copies of the synthetic E. coli rRNA target.

Example 2

THE USE OF A MARKED PRIMING OLIGONUCLEOTIDE ALLOWS THE DISTINCTION BETWEEN SAMPLE MOLDS AND EXOGENOUS MOLDS

A. AMPLIFICATION BY USING AN UNBAGED PRIMING OLIGONUCLEOTIDE WITHOUT TARGET CAPTURE

In a first procedure, amplification reactions employing a synthetic E. coli rRNA template were performed using an unlabeled primer oligonucleotide that hybridized to the template, a promoter oligonucleotide, a terminator oligonucleotide, and a probe probe. molecular torch detection. Reactions were primed using the synthetic template added directly to the reaction mixtures (i.e. without performing target capture purification) at 0 or 10⁶ copies / reaction. A molecular torch detection probe was used to monitor amplicon production as a function of time. In the nucleotide sequences presented below, 2'-O-methyl ribose (2'-O-Me) modifications of the polynucleotide backbone are indicated in lower case, m. The blocking residues at the 3 'ends of the promoter oligonucleotide and terminator oligonucleotide consisted of a 3'-a-3 linkage that was prepared using 3'-dimethyltrityl-N-benzoyl-2'-deoxycytidine, 5'-succinoyl -long chain alkylamino-CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). The essential oligonucleotides, reagents and methods used in the procedure were as follows.

I. OLIGONUCLEOTIDES:

1. Unlabeled Priming Oligonucleotide:

CmUmGmCmTGGCACGGAGTTAGCCGGTGCTTC (SEQ ID NO: 9)

2. Promoter Oligonucleotide:

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG - blocking (SEQ ID NO: 10)

3. Termination Oligonucleotide:

GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - lock (SEQ ID NO: 11)

Four. Detection Probe:

¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCmAmAmAm²CGCAG³(SEQ ID NO: 12)

ES 2 358 296 T3 ¹ fluorescein ² C9 spacer ³ DABCYL

5. Synthetic E. coli rRNA template:

AAATTGAAGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCC TAACACATGCAAGTCGAACGGTAACAGGAAGAAGCTTGCTTCTTTGCTGACGA GTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAAC TACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGA CCTrCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGG 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:

The amplification and enzyme reagents were essentially as described in Example 1. Procedures using the unlabeled primer oligonucleotide that hybridized to the E. coli template did not use target capture reagents or oligonucleotides, did not use transport medium. or wash solution, and did not use an elongated oligonucleotide.

A. Real Time Amplification Protocol.

Sample solutions were prepared using a primerless amplification reagent, unlabeled primer oligonucleotide, promoter oligonucleotide, terminator oligonucleotide, detection probe, and synthetic template nucleic acid. Each well of a 96-well microtiter plate received a 30 pL aliquot of the prepared sample solution. The microtiter plate was covered with an adhesive tape closure, first incubated for 10 minutes at 60 ° C in the DNA ENGINE OPTICON® 2 real-time temperature controlled instrument (Bio-Rad Laboratories; Hercules, CA), and then the temperature was adjusted to 42 ° C for 5 minutes. The plate was then removed from the real-time instrument and placed in a thermomixer at 42 ° C. Each reaction well received a 10 pL aliquot of the enzyme reagent. The microtiter plate was covered with an adhesive tape closure, gently shaken for 30 seconds in the thermomixer, and then placed in the real-time instrument at 42 ° C, where real-time assay monitoring began. The TTtime values, which served as indicators of the amount of amplicon synthesized, were determined from the monitored fluorescence signals.

ES 2 358 296 T3

III. RESULTS AND CONCLUSION

As indicated in Figure 20, substantially identical results were observed in reactions that included 0 or 10⁶ copies of the template nucleic acid, and thus the assay showed no distinction between these two conditions. More specifically, fluorescent signals indicating the formation of E. coli nucleic acid amplification products emerged from background levels at substantially similar times (i.e., Ttime = 31.74 minutes at the 0 copy level, and 31 , 19 minutes at level 10⁶ copies) in both reactions. Thus, a characteristic real-time amplification profile of high levels of nucleic acid template was obtained even in the absence of rRNA template of

E. coli added. This was consistent with the presence of contaminating bacterial nucleic acid templates in one or more of the reagents used to carry out the amplification reactions after the target capture procedure.

B. AMPLIFICATION THROUGH THE USE OF A MARKED PRIME OLIGONUCLEOTIDE AND TARGET CAPTURE

In a second procedure, a labeled priming oligonucleotide and a target capture step were employed to carry out the amplification reactions using the test samples containing 0.10³ or 10⁵ copies of the synthetic E. coli transcript. The oligonucleotides used in the procedure are listed below. The molecular torch detection probe was added as a component of the enzyme reagent. Following target capture, the labeled primer oligonucleotide that was not hybridized to the template nucleic acid was removed from the system by standard wash and target capture steps. The complex that included the rRNA template and the labeled primer oligonucleotide remained captured on the super-paramagnetic particles. Amplification reactions were carried out using reagents essentially as described above, except for the substitution of a non-specific target capture probe for the sequence-specific capture probes employed in Example 1. Amplification reactions were carried out in replicates of six and were monitored using a molecular torch detection probe essentially as described in Example 1, except that the elongating oligonucleotide was omitted. As before, 2'-O-methyl ribose (2'-O-Me) modifications of the polynucleotide backbone in the sequences presented below are indicated by the lowercase letter m. The blocking residues at the 3 'ends of the promoter oligonucleotide and terminator oligonucleotide consisted of a 3'-to-3' bond that was prepared using 3'-dimethyltrityl-N-benzoyl-2'deoxycytidine, 5'-succinoyl -long chain alkylamino-CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). The essential oligonucleotides, reagents and methods used in the procedure were as follows.

I. Oligonucleotides:

1. Marked Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCG GTGCTTC (SEQ ID NO: 14)

2. Marker Specific Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter Oligonucleotide:

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG -locking (SEQ ID NO: 10)

Four. Termination Oligonucleotide:

GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - lock (SEQ ID NO: 11)

5. Nonspecific Capture Sonar:

KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTTAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA (SEQ ID NO: 16)

6. Detection Probe:

¹CmUmGmCmGmGmGmUmAmAmCmGmUmCmAmAmUmGmAmGmCmAmAmAm²CGCAG³(SEQ ID NO: 12) ¹ fluorescein ² C9 spacer

ES 2 358 296 T3 ³ DABCYL

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

II. REAGENTS AND OTHER PROTOCOL INFORMATION

The experimental reagents and protocols were essentially as described in Example 1, with the substitution of a non-specific target capture oligonucleotide for the first and second capture oligonucleotides, the replacement of the E. coli-specific oligonucleotides presented above by the HCV-specific oligonucleotides, and the omission of a lengthening oligonucleotide.

III. NON-SPECIFIC TARGET CAPTURE PROTOCOL

A. Preparation of the Target Capture Reagent (TCR).

A stock suspension of magnetic spheres was mixed at room temperature for 30 minutes. An aliquot of about 150 pL of the magnetic bead suspension was added to 5 mL of TCR solvent (15 pg of beads / reaction using 50 pL / sample), and then slowly mixed at room temperature for 30 minutes. The nonspecific capture oligonucleotide was then added to 5 mL of the TCR mixture to provide a final concentration of 0.12 pmol / pL. The prepared TCR was mixed gently at room temperature until needed.

B. Sample Preparation.

The amplification solution was prepared using primerless amplification reagent, promoter oligonucleotide, and marker specific priming oligonucleotide. The prepared amplification solution was mixed by vortexing and holding 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. Dilutions of the template rRNA were prepared in 0.2% LLS (lithium lauryl sulfate). Aliquots (50 pL) of the target capture solution with magnetic beads were transferred to the wells of a microtiter plate for a KINGFISHER 96 magnetic particle processor (Thermo Fisher Scientific, Inc .; Waltham, MA). Samples of diluted template, labeled primer oligonucleotide, and terminator oligonucleotide were then added to 1.5 mL of transport medium diluted 50% with water. The sample mixture containing the target was vortexed, and 150 pL aliquots were transferred to the wells of the microtiter plate (Plate 1) containing 50 pL of target capture solution (each well contained 0.10³ or 10⁵ copies of the E. coli transcript and the appropriate amount of labeled primer oligonucleotide and terminator oligonucleotide).

C. Target Capture Protocol.

First, a microtiter plate was prepared containing 200 pL of wash reagent (Plate 2). Another microtiter plate (Plate 3) was prepared to carry out the amplification reactions, and each well to be used for a reaction contained 30 pL of amplification reagent. All three plates (Plates 1-3) were loaded into the magnetic particle processor unit. Magnetic particles harboring nucleic acid complexes were isolated from Plate 1, washed on Plate 2, and then transferred to Plate 3 using standard procedures familiar to those of ordinary skill level in the art. . Plate 3 was removed from the magnetic particle processing unit, covered with an adhesive tape closure, and then placed in the temperature controlled real-time instrument.

D. Real Time Amplification Protocol.

Plate 3 was incubated at 42 ° C for 5 minutes on the real time instrument. The microtiter plate was removed from the real time instrument and placed in a thermomixer at 42 ° C. Each reaction well received a 10 pL aliquot of the enzyme reagent containing the detection probe, and was then covered with an adhesive tape closure. The plate was gently shaken for 60 seconds on the thermomixer, and then returned to the 42 ° C real-time instrument, where real-time assay monitoring began. The TTtime values, which served as indicators of the amount of amplicon synthesized, were determined from the monitored fluorescence signals.

IV. RESULTS AND CONCLUSION

Figure 21 graphically illustrates the benefits of the described approach for nucleic acid amplification. Procedures employing a labeled primer oligonucleotide complementary to a target of interest, a target capture step, and a marker-specific primer oligonucleotide that was not complementary to the target of interest (i.e., the E.

ES 2 358 296 T3 coli) produced very low background amplification levels, thus allowing the easy distinction between 0 and 10³ copies of the bacterial template nucleic acid. More specifically, the mean time values ​​determined for reactions carried out using 10⁵ copies, 10³copies, and 0 copies of the E. coli template were 24.7 minutes, 30.6 minutes, and 37.5 minutes, respectively. Considered in conjunction with the results presented in Figure 4, these findings were consistent with the presence of nucleic acids derived from bacteria in common reagents used to carry out nucleic acid amplification reactions in vitro. Despite this fact, the method employing a labeled primer oligonucleotide was useful to detect E. coli nucleic acids contained in a test sample without interference from exogenous template nucleic acids contributed by the amplification reagents. For example, a qualitative assay to detect E. coli nucleic acids at a level of 10³ copies or more in a test sample could be dependent on reaching a threshold fluorescence signal, or a value of TTime after a predetermined reaction time (eg, 35 minutes).

*****

The following Example presents comparative results showing how two different detection probes influenced the profiles of real-time amplification curves. The results further demonstrated how the labeled priming oligonucleotide approximation could be used to distinguish between 0 and 10.³ copies of the synthetic E. coli template nucleic acid, a level that approximates the number of copies of 16S rRNA present in a single bacterium.

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

Example 3

ALTERNATIVE TORCH DESIGNS MAY IMPROVE TEST RESULTS

Amplification reactions were carried out and monitored in a real time format using one to three different detection probes. The synthetic template nucleic acid, nonspecific capture oligonucleotide, labeled primer oligonucleotide, terminator oligonucleotide, promoter oligonucleotide, and marker specific primer oligonucleotide used to perform the reactions were identical to those used in the second procedure. of the preceding Example. The target hybridization portion of E. coli of the labeled primer oligonucleotide corresponded to nucleotide positions 24-57 of SEQ ID NO: 14 (ie, the target hybridization sequence corresponded to SEQ ID NO: 19). The E. coli target hybridization portion of the promoter oligonucleotide corresponded to nucleotide positions 27-47 of SEQ ID NO: 10 (ie, the target hybridization sequence corresponded to SEQ ID NO: 20). Four replicates were used for each condition. As before, the detection probe was added with the enzyme reagent. The reagents and protocols for nonspecific target capture, sample preparation, and real-time amplification were also essentially as described in the second procedure of the preceding Example. Notably, the reactions were carried out using 0.10³ or 10⁵ copies of the synthetic E. coli template. As before, 2'-Omethyl ribose (OMe) modifications of the polynucleotide backbone in the sequences presented below are indicated by the lowercase letter m. The blocking residues at the 3 'ends of the promoter oligonucleotide and terminator oligonucleotide consisted of a 3'-to-3' junction that was prepared using 3'-dimethyltrityl-N-benzoyl-2'-deoxycytidine, 5'- succinoyl-long chain alkylamino-CPG (Glen Research Corporation, Sterling, VA; Cat. No. 20-0102-01). The essential oligonucleotides, reagents and methods used in the procedure were as follows.

I. OLIGONUCLEOTIDES:

1. Marked Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTATGTCTACCTGCTGGCACGGAGTTAGCCG GTGCTTC (SEQ ID NO: 14)

2. Marker Specific Priming Oligonucleotide:

GTTTGTATGTCTGTTGCTATTAT (SEQ ID NO: 15)

3. Promoter Oligonucleotide:

ATTTAATACGACTCACTATAGGGAGAGAAGGCCTTCGGGTTGTAAAG - blocking (SEQ ID NO: 10)

Four. Termination Oligonucleotide:

ES 2 358 296 T3

GmCmCmUmUmCmUmUmCmAmUmAmCmAmCmGmCmGm - lock (SEQ ID NO: 11)

5. Nonspecific Capture Sonar:

KmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmKmTTTAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA (SEQ ID NO: 16)

6. Detection Probe:

¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCm²GmCmUmCmGm³ (SEQ ID NO: 17) ¹CmGmAmGmCmAmAmAmGmGmUmAmUmUmAmAmCmUmUmUmAmCmUmCm²GmCmUmC mGm³ (SEQ ID NO: 18) ¹ fluorescein ² C9 spacer ³ DABCYL

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

II. REAGENTS AND OTHER PROTOCOL INFORMATION

The reagents and experimental protocols were essentially as described in Example 2, with a slight change in the conditions used for target capture.

III. NON-SPECIFIC TARGET CAPTURE PROTOCOL:

A. Preparation of the Target Capture Reagent (TCR).

A stock suspension of magnetic spheres was mixed at room temperature for 25 minutes. A 150 pL aliquot of the magnetic bead suspension was added to 5 mL of TCR solvent (15 pg spheres / reaction using 50 pL / sample), and then slowly mixed at room temperature for 25 minutes. The nonspecific capture oligonucleotide was then added to 5 mL of the TCR mixture to provide a final concentration of 0.12 pmol / pL. The prepared TCR was mixed gently at room temperature until needed.

B. Sample Preparation.

Amplification solutions were prepared using AMP Reagent without primer, promoter oligonucleotide, and marker specific priming oligonucleotide. The prepared amplification solutions were mixed by vortexing and then kept at 2-8 ° C until needed. Enzyme Reagents containing molecular torch detection probes were then prepared and kept at 2-8 ° C until needed. Dilutions of the template rRNA were prepared in 0.2% LLS, as described above. Aliquots (50 pL) of the target capture solution with magnetic beads were transferred to the wells of a microtiter plate for a KINGFISHER 96 magnetic particle processor (Thermo Fisher Scientific, Inc .; Waltham, MA). Samples of diluted template, labeled primer oligonucleotide, and terminator oligonucleotide were then added to 1.5 mL of Transport Medium diluted 50% with water. The sample mixture containing target was vortexed, and 150 pL aliquots were transferred to the wells of the microtiter plate (Plate 1) containing 50 pL of target capture solution (each well contained 0.10³ or 10⁵ E. coli transcript copies and the appropriate amount of labeled primer oligonucleotide and terminator oligonucleotide).

C. Target Capture Protocol.

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). Next, a second microtiter plate was prepared containing 200 pL of Wash Reagent (Plate 2). A third microtiter plate (Plate 3) was prepared to carry out the amplification reactions, and each well to be used for a reaction contained 30 pL of amplification reagent. All three plates were placed in the magnetic particle processing unit. Magnetic spheres harboring nucleic acid complexes were isolated from Plate 1, washed on Plate 2, and then transferred to Plate 3 using standard procedures familiar to those of ordinary skill in the art. Plate 3 was removed from the particle processor unit

ES 2 358 296 T3, was covered with an adhesive tape closure, and then placed on the temperature-controlled real-time instrument.

D. Real Time Amplification Protocol.

Plate 3 was incubated in the real time instrument at 42 ° C for 5 minutes. The microtiter plate was removed from the real time instrument and placed in the thermomixer at 42 ° C. Each reaction well received a 10 pL aliquot of Enzyme Reagent containing the detection probe, and was then covered with an adhesive tape closure. The plate was gently shaken for 60 seconds on the thermomixer, and then returned to the 42 ° C real-time instrument, where real-time assay monitoring began. The TTtime values, which served as indicators of the amount of amplicon synthesized, were determined from the monitored fluorescence signals.

IV. RESULTS AND CONCLUSION

The results presented in Table 2 summarize the mean TTtime values ​​(column 3), and the standard deviations of the mean TTtime values ​​(column 4) for the reactions carried out using the different detection probes. The tabular summary confirmed that all detection probes tested gave very good results in real time assays. Each probe advantageously provided a very low signal at the initial target 0 copy level. More specifically, the amplicon detected in reactions carried out using 0 copies of initial synthetic template was essentially undetectable when the reactions included the detection probes of SEQ ID NO: 17 and SEQ ID NO: 18. Thus, reactions that included one of the detection probes identified by SEQ ID NO: 17 and SEQ ID NO: 18 provided extraordinary results that easily allowed the detection of template nucleic acids that roughly corresponded to the amount contained in a single bacterium.

Table 2. Use of Alternative Detection Probes for Enhanced Assay Distinction

<td>Mold Quantity (copies)</td><td>Detection Probe</td><td>Average time (minutes)</td><td>Time DE (minutes)</td>

<td> 0</td><td></td><td>N / A</td><td>N / A</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>N / A</td><td>N / A</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>

In view of the results presented in Examples 2 and 3, each of SEQ ID NOs: 12, and 17-18 represent preferred molecular torches for the detection of E. coli using the methods described herein. The most preferred probes useful for the detection of E. coli will have sequences complementary to the target corresponding to nucleotide positions 2-24 contained in the probe of SEQ ID NO: 12 (i.e., the target hybridization sequence corresponding to SEQ ID NO: 21), or positions of nucleotides 2-17 contained in the probe of SEQ ID NO: 17 (i.e., the target hybridization sequence corresponding to SEQ ID NO: 22), or the positions of nucleotides 2-24 contained in the probe of SEQ ID NO: 18 (i.e. the hybridization sequence to the target corresponding to SEQ ID NO: 23). In general, probes useful for the detection of E. coli nucleic acids will have target hybridization sequences of at least 16 contiguous nucleotides contained within the sequence of TGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTC (SEQ ID NO: 24). Overall preferred lengths of the desirable 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 can include RNA and DNA equivalent bases, and include complements to the probes described above.

ES 2 358 296 T3

SEQUENCE LIST &lt;110&gt; GEN-PROBE INCORPORATED

BECKER, Michael M.

LIVEZEY, Kristin W.

LAM, Wai-Chung W.

&lt;120&gt; MARKED OLIGONUCLEOTIDES AND THE USE OF THEM IN NUCLEIC ACID AMPLIFICATION METHODS &lt;130&gt; GP193-PCT &lt;140&gt; Will be assigned &lt;141&gt; 2007-06-06 &lt;150&gt; 60 / 871,442 &lt;151&gt; 2006-12-21 &lt;150&gt; 60 / 811,581 &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; HCV-specific, labeled 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 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Marker-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;

ES 2 358 296 T3 &lt;223&gt; HCV specific promoter oligonucleotide &lt;400&gt; 3 atttaatacg actcactata gggagaccac aacggtttct agccatggcg ttagtatgag 60 &lt;210&gt; 4 &lt;211&gt; 26 &lt;212&gt; RNA &lt;213&gt; Hepatitis C virus &lt;220&gt;

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (1) .. (26) &lt;223&gt; 2 'methoxy analogs &lt;400&gt; 4 auggcuagac gcuuucugcg ugaaga &lt;210&gt; 5 &lt;211&gt; 42 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; Extender oligonucleotide &lt;400&gt; 5 tgtcgtgcag cctccaggac cccccctccc gggagagcca ta &lt;210&gt; 6 &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; Artificial &lt;220&gt;

&lt;223&gt; HCV-specific capture probe &lt;400&gt; 7 cauggugcac ggucuacgtt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa a

ES 2 358 296 T3 &lt;210&gt; 8 &lt;211&gt; 23 &lt;212&gt; RNA &lt;213&gt; Artificial &lt;220&gt;

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

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (1) .. (23) &lt;223&gt; 2 'methoxy analogues &lt;220&gt;

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

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (1) .. (4) &lt;223&gt; 2 'methoxy analogs &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 &lt;210&gt; 11 &lt;211&gt; 18 &lt;212&gt; RNA

ES 2 358 296 T3 &lt;213&gt; Escherichia coli &lt;220&gt;

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

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

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

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (24) .. (25) &lt;223&gt; non-nucleotide bond &lt;220&gt;

&lt;221&gt; miscellaneous characteristic &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 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic E. coli rRNA template &lt;400&gt; 13

ES 2 358 296 T3

<td>aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa</td><td> 60</td>

<td>gtcgaacggt aacaggaaga agcttgcttc tttgctgacg agtggcggac gggtgagtaa</td><td> 120</td>

<td>tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct aataccgcat</td><td> 180</td>

<td>aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg tgcccagatg</td><td> 240</td>

<td>ggattagcta gtaggtgggg taacggctca cctaggcgac gatccctagc tggtctgaga</td><td> 300</td>

<td>ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag gcagcagtgg</td><td> 360</td>

<td>ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg aagaaggcct</td><td> 420</td>

<td>tcgggttgta aagtactttc agcggggagg aagggagtaa agttaatacc tttgctcatt</td><td> 480</td>

<td>gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag</td><td> 540</td>

<td>ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt tgttaagtca</td><td> 600</td>

<td>gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag cttgagtctc</td><td> 660</td>

<td>gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc</td><td> 720</td>

<td>ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca</td><td> 780</td>

<td>aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc</td><td> 840</td>

<td>cttgaggcgt ggcttccgga gctaacgcgt taagtcgacc gcctggggag tacggccgca</td><td> 900</td>

<td>aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat</td><td> 960</td>

<td>tcgatgcaac gcgaagaacc ttacctggtc ttgacatcca cggaagtttt cagagatgag</td><td> 1020</td>

<td>aatgtgcctt cgggaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga</td><td> 1080</td>

<td>aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc</td><td> 1140</td>

<td>cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc</td><td> 1200</td>

<td>atcatggccc ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg</td><td> 1260</td>

<td>acctcgcgag agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac</td><td> 1320</td>

<td>tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt</td><td> 1380</td>

<td>tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt</td><td> 1440</td>

<td>agcttaacct tcgggagggc gcttaccact ttgtgattca tgactggggt gaagtcgtaa</td><td> 1500</td>

<td>caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta</td><td> 1542</td>

&lt;210&gt; 14 &lt;211&gt; 57 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; E. coli-specific labeled priming oligonucleotide &lt;400&gt; 14 gtttgtatgt ctgttgctat tatgtctacc tgctggcacg gagttagccg gtgcttc 57 &lt;210&gt; 15

ES 2 358 296 T3 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; marker 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; miscellaneous characteristic &lt;222&gt; (1) .. (18) &lt;223&gt; 2 'methoxy analogs &lt;220&gt;

&lt;221&gt; miscellaneous feature &lt;222&gt; (19) .. (51) &lt;223&gt; DNA &lt;400&gt; 16 kkkkkkkkkk kkkkkkkktt taaaaaaaaa aaaaaaaaaa aaaaaaaaaa to &lt;210&gt; 17 &lt;211&gt; 22 &lt;212&gt; RNA2 &lt;213&gt; Artificial

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

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

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (17) .. (18) &lt;223&gt; non-nucleotide bond

ES 2 358 296 T3 &lt;400&gt; 17 cgagcaaagg uauuaacgcu cg &lt;210&gt; 18 &lt;211&gt; 29 &lt;212&gt; RNA &lt;213&gt; Artificial &lt;220&gt;

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

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

&lt;221&gt; miscellaneous characteristic &lt;222&gt; (24) .. (25) &lt;223&gt; non-nucleotide bond &lt;400&gt; 18 cgagcaaagg uauuaacuuu acucgcucg 29 &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; Escherichia coli &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

ES 2 358 296 T3

<td colspan="5"> &lt;211&gt; 16</td>

<td></td><td>&lt;212&gt; RNA &lt;213&gt; Escherichia coli &lt;400&gt; 22</td><td></td><td></td><td></td>

<td> 5</td><td>gagcaaaggu auuaac &lt;210&gt; 23 &lt;211&gt; 23 &lt;212&gt; RNA &lt;213&gt; Escherichia coli</td><td></td><td></td><td> 16</td>

<td> 10</td><td>&lt;400&gt; 23 gagcaaaggu auuaacuuua cuc &lt;210&gt; 24 &lt;211&gt; 39 &lt;212&gt; DNA</td><td></td><td> 23</td><td></td>

<td> 15</td><td>&lt;213&gt; Escherichia coli &lt;400&gt; 24 tgcgggtaac gtcaatgagc aaaggtatta actttactc</td><td> 39</td><td></td><td></td>