Nucleic acid sequences that can be used as primers and probes in the amplification and detection of all subtypes of hiv-1

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DESCRIPTION

Nucleic acid sequences that can be used as primers and probes in the amplification and detection of all HIV-1 subtypes.

The present invention relates to nucleic acid sequences that can be used in the field of virus diagnosis, more specifically in the diagnosis of infections with the Human Immunodeficiency Virus (HIV) that causes AIDS.

Although conventional virus diagnosis has been predominantly based on the detection of viral antigens or antibodies specific to them, in recent years attention has shifted towards methods for the direct detection of the virus genome or nucleic acid sequences derived from it. , both RNA and DNA. These methods are generally based on nucleic acid hybridization. Nucleic acid hybridization relies on the ability of two nucleic acid strands containing complementary sequences to anneal to each other under appropriate conditions, thus forming a double braided structure. When the complementary strand is tagged, the tag can be detected and is indicative of the presence of the target sequence. Especially in combination with methods for nucleic acid sequence amplification, these methods have become an important viral diagnostic tool, in particular for the detection of human immunodeficiency virus (HIV).

Nucleic acid amplification techniques are especially useful as an additional technique in cases where serological methods give questionable results or in cases where there may be a considerable period of time between infection and the development of antibodies to it. virus. With HIV, seroconversion can usually occur about 3 to 6 months after exposure to the virus. Thus, although antibodies are not detected with conventional immunoassays, circulating viral RNA or proviral DNA may be detectable. Also, in follow-up antiviral therapy, nucleic acid amplification-based methods have several advantages over serological methods. Especially quantitative amplification methods provide a powerful tool for assessing changes in the amount of virus present before and during therapy.

The choice of oligonucleotides to be used as primers and probes in the amplification and detection of nucleic acid sequences is critical to the sensitivity and specificity of the assay. Normally, the sequence to be enlarged is present only in a sample (for example in a blood sample obtained from a patient who is supposedly suffering from the viral infection) in minute quantities. The primers should be sufficiently complementary to the target sequence to allow efficient amplification of the viral nucleic acid present in the sample. If the primers are not annealed properly (due to poor nucleotide base pairing on both strands) to the target sequence, amplification will be seriously impaired. This will affect the sensitivity of the assay and may result in false negative assay results. Due to the heterogeneity of viral genomes, false negative test results can be obtained when the primers and probes are able to recognize the sequences present only in part of the virus variants. The HIV virus is highly heterogeneous. Genetic variability has been demonstrated between isolates from different continents, but also between different individuals and between different stages of the disease. Based on sequence analysis, two groups have been identified within HIV-1: group M (M for "major" - major), and group O (O for annexed outlier). Within group M, subtypes (HA) have been assigned, each constituting a separate group of phylogenetic sequences, and additional ones are being identified. This sequence variation is not uniformly distributed throughout the genome. The HIV1 genome, like all retroviral genomes, roughly consists of the following regions: the gag gene of the HIV-1 genome is the region that encodes the core proteins of the virus (eg, p24); the env gene encodes a large precursor protein, gp 160, which is processed to become the envelope proteins gp 120 and gp41. The pol gene encodes the polymerase of the virus (reverse transcriptase). The Long Terminal Repeat (LTR) regions are the regions in the viral genome that participate in the integration of the virus with the host cell and in the regulation of the transcription of viral genes. Some regions are more prone to sequence variation than others. Above all, in the env domain sequence, the variation can reach up to 30% between members of different subtypes. Ideally, primer selection should be based on information about strain-to-strain variability in candidate primer sequences and the consequences of poor primer site matching. McCutcheon et al., J. AIDS, 4, 1241-1250, 1991, use PCR to perform a genetic comparison of different HIV-1 isolates. Using tethered PCR (multi-sense primers with a constant antisense primer were used; primers from relatively conserved regions in gag, env and LTR were selected). The effect of primer mismatch on the amount of PCR product obtained was investigated. Poor matching at the 3 'end of the primer decreased the amount of product sometimes by more than 100 times.

The detection of all currently known HIV-1 subtypes is of enormous importance, especially in terms of patient management, the safety of blood and blood products, and clinical and epidemiological studies. Current assays for the amplification and subsequent detection of HIV-1 derived nucleic acid sequences are typically based on the amplification of sequences in the gag region of the viral genome. These assays have been developed to detect subtype B, which is the main subtype in European countries and in the United States. However, the presence of other subtypes, which were previously geographically confined, is increasing due to frequent movements between these countries and, for example, African countries. For the

Therefore, sensitive assays are needed that can detect as many HIV-1 virus variants as possible (preferably all).

Research aimed at identifying suitable primer sets for reliable amplification of HIV-1 derived nucleic acid sequences has been ongoing for the past years. Engelbrecht et al., J. Virol. Meth., 55, 391-400, 1995, describe a study aimed at the development of a specific and sensitive PCR protocol using env, gag and LTR primer pairs to detect subtypes present in the Western Cape, South Africa. Twenty-four strains known to belong to subtypes B, C, and D were tested. The performance of the primer pairs was found to be highly dependent on the optimization of the reaction conditions for the different primer pairs. Only when less stringent conditions were used (e.g. with the LTR primer pair, increased cycle time and lower anelation temperatures were required) could these particular HIV strains be detected with sufficient sensitivity and reproducibility with all pairs of primer.

Zazzi et al. J. Med. Virol., 38, 172-174, 1992, developed a two-step PCR reaction (using nested primers) to detect HIV-1 DNA in clinical samples. The primers used for amplification were derived from the gag gene and the LTR region. The patients tested in this study were all from neighboring areas, making it likely that they represented only a limited number of different viral strains. In EPA-0.617.132; US-5,629,413 and EP-A-0,331,939 set forth other examples of PCR primers for the detection of HIV.

Vener et al., In Bio Techniques, 21, 248-255, 1996 describe a quantitative PCR method using nested primers derived from LTRs. This procedure was tested only in peripheral blood mononuclear cells (PBMC), infected with HIV-1_MN. Therefore, nothing can be said about the suitability of the primers used for the detection of different virus subtypes.

With the present invention, nucleotide sequences are provided that can be used as primers and probes in the amplification and detection of HIV-1 nucleic acid. The oligonucleotide sequences provided by the present invention are located in the LTR part of the HIV viral genome. It has been found that by using the sequences of the present invention in methods for nucleic acid amplification and detection, sensitive and specific detection of HIV-1 can be obtained. The benefit of the sequences of the present invention resides mainly in the fact that, with the help of primers and probes comprising the sequences according to the invention, the nucleic acid of all currently known HIV-1 subtypes can be detected, with high precision and sensitivity. Until now, no hybridization primer or probe pair has been developed that would allow the detection of such a wide range of HIV-1 variants.

The oligonucleotide sequences according to the present invention are especially useful in nucleic acid amplification methods.

Various techniques for nucleic acid amplification are known in the art. An example of a technique for amplifying a target DNA segment is the so-called "polymerase chain reaction" (PCR). With the PCR technique, the copy number of a particular target segment is exponentially increased with a series of cycles. A pair of primers is used and, in each cycle, a DNA primer is annotated with the 3 'side of each of the two strands of the target double-stranded DNA sequence. The primers are extended with a DNA polymerase in the presence of the various mononucleotides to generate double-stranded DNA again. The double stranded DNA strands are separated from each other by thermal denaturation and each strand serves as a template for primer annealing and subsequent elongation in a subsequent cycle. The PCR method has been described by Saiki et al., Science 230, 135, 1985, and in European patents No. EP 200362 and EP 201184.

Another technique for nucleic acid amplification is the so-called transcription-based amplification system (TAS). The TAS method is described in International Patent Application No. WO 88/10315. Transcription-based amplification techniques typically include treating the target nucleic acid with two oligonucleotides, one including a promoter sequence, to generate a template that includes a functional promoter. Multiple copies of RNA are transcribed from said template that can serve as the basis for further amplification.

An amplification method based on isothermal continuous transcription is the so-called NASBA process ("NASBA"), as described in European Patent No. EP 329822. NASBA includes the use of T7 RNA polymerase to transcribe multiple copies of RNA from of a template that includes a T7 promoter. Other transcription-based amplification techniques are described in EP 408295. EP 408295 is primarily concerned with an amplification method based on the transcription of two enzymes. Transcription-based amplification methods, for example the NASBA method described in EP 329822, are normally used with a group of oligonucleotides, in one of them providing a promoter sequence that is recognized by a DNA-dependent RNA polymerase such as, for example T7 polymerase. Various modifications of transcription-based techniques are known within the art. Such modifications include for example the use of blocked oligonucleotides (into which a promoter sequence can be incorporated). These oligonucleotides are blocked to inhibit an extension reaction from them (US5554516). One or more "promoter primers" (oligonucleotides equipped with a promoter sequence) can be used in transcription-based amplification techniques, optionally combined with the use of one or more oligonucleotides that are not equipped with a promoter sequence. For the

ES 2 153 807 T3 RNA amplification, the preferable technology is a transcription-based amplification technique. Amplification using PCR can also be based on an RNA template. The actual PCR requires to be preceded by a reverse transcription step to copy the RNA into DNA (RT-PCR). However, if RT-PCR is used for the detection of viral transcripts, differentiation of mRNA and DNA derived PCR products is necessary. DNAse treatment can be applied before RT-PCR (Bitsch, A. et al., J. Infect. Dis 167, 740-743., 1993; Meyer, T. et al., Mol. Cell Probes. 8, 261-271, 1994), but is sometimes not efficient at removing contaminating DNA sufficiently (Bitsch, A. et al., 1993).

In contrast to RT-PCR, NASBA, which is based on RNA transcription by T7 RNA polymerase (Kievits et al., 1991; Compton, 1991) does not require differentiation between RNA and DNA-derived amplification products since it uses RNA as its main target. NASBA enables specific amplification of RNA targets even in a DNA environment.

The use of the oligonucleotides according to the invention is not limited to any particular amplification technique or specific modification thereof. It is clear that the oligonucleotides according to the invention find application in many different nucleic acid amplification techniques, as well as various methods for detecting the presence of HIV (amplified) nucleic acid. The oligonucleotides of the present invention can also be used in quantitative amplification methods. An example of such a quantitative method is described in EP 525882.

The term "oligonucleotide" as used herein refers to a molecule consisting of two or more deoxyribonucleotides or ribonucleotides. Said oligonucleotides can be used as primers and probes.

Naturally, based on the sequences of the oligonucleotides of the present invention, oligonucleotide analogs can also be prepared. Such analogs can constitute alternative structures such as "PNAs" (molecules with a peptide-like structure instead of the phosphate sugar structure of normal nucleic acid) or the like. It is clear that these alternative structures, which represent the sequences of the present invention, form part of the present invention as well.

The term "primer" as used herein refers to an oligonucleotide that occurs either naturally (eg, as a restriction fragment) or synthetically produced, that is capable of acting as a starting point for the synthesis of a product. primer extension that is complementary to the nucleic acid strand (template or target sequence) when placed under the proper conditions (eg, buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization, such as RNA-dependent or DNA-dependent polymerase. A primer must be long enough to prime the synthesis of extension products in the presence of an agent for polymerization. A typical primer contains at least about 10 nucleotides in length of a sequence substantially complementary or homologous to the target sequence, although somewhat longer primers are preferred. Primers typically contain about 15-26 nucleotides, although longer primers can be used, especially when the primers contain additional sequences such as a promoter sequence for a particular polymerase.

Typically, a set of primers will consist of at least two primers, one "upstream" and one "downstream" that together define the amplifier (the sequence that will be amplified using such primers).

Primarily, for use in transcription-based amplification techniques, the oligonucleotides according to the invention can also be linked to a promoter sequence. The term "promoter sequence" defines a region of a nucleic acid sequence that is specifically recognized by an RNA polymerase that binds to a recognized sequence and initiates the transcriptional process through which the RNA transcript is produced. In principle, any promoter sequence for which a polymerase is known and available which is capable of recognizing the initiation sequence can be used. Known and useful promoters are those that are recognized by certain bacteriophage RNA polymerases such as T3, T7 or SP6 bactariophages. Oligonucleotides attached to a promoter sequence are commonly referred to as "promoter primers." However, its function as a primer, eg, the starting point for an elongation reaction, can be blocked, as mentioned above, or absent in some embodiments of transcription-based amplification reactions.

An oligonucleotide according to the present invention is substantially complementary to a sequence of the LTR region of a nucleic acid sequence of an HIV genome, said oligonucleotide being 10-50 nucleotides in length and comprising, at least one 10 nucleotide fragment, of a sequence selected from the group consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA

SEQ ID 2: G TTC GGG CGC CAC TGC TAG A

SEQ ID 3: CGGGCGCCACTGCTA

SEQ ID 4: CTG CTT AAA GCC TCA ATA AA

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SEQ ID 5: CTC AAT AAA GCT TGC CTT GA

SEQ ID 6: TCT GGT AAC TAG AGA TCC CTC

SEQ ID 7: TAG TGT GTG CCC GTC TGT

SEQ ID 8: AGT GTG TGC CCG TCT GTT

SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT or complementary sequence thereof.

It should be understood that the oligonucleotides consisting of the sequences of the present invention may contain minor deletions, additions and / or substitutions of nucleic acid bases, to an extent that such alterations do not adversely affect the yield of the obtained product to any significant degree. . When the oligonucleotides according to the present invention are used as probes, the alterations should not result in reduced hybridization efficiency of the probe. For example, in the case of amplification techniques based on transcription, in which a promoter sequence can be incorporated into one or more of the primers, the introduction of a hybridization sequence rich in purine (= G or A), immediately after the promoter sequence, it can provide a positive effect on transcription (when there are C's and T's, abortive transcription can occur). If no such sequence is available in the target nucleic acid, a purine-rich sequence can be inserted into the oligonucleotide immediately after the last three G radicals of the promoter sequence. The sequences of the present invention are reflected as DNA sequences. The RNA equivalents of these sequences also form part of the present invention.

Preferable oligonucleotides according to the present invention are oligonucleotides consisting essentially of a sequence selected from the group consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA

SEQ ID 2: G TTC GGG CGC CAC TGC TAG A

SEQ ID 3: CGGGCGCCACTGCTA

SEQ ID 4: CTG CTT AAA GCC TCA ATA AA

SEQ ID 5: CTC AAT AAA GCT TGC CTT GA

SEQ ID 6: TCT GGT AAC TAG AGA TCC CTC

SEQ ID 7: TAG TGT GTG CCC GTC TGT

SEQ ID 8: AGT GTG TGC CCG TCT GTT

SEQ ID 9: aat tct aat acg act cac tat agg gAG AGG GGC GCC ACT GCT AGA GA

SEQ ID 10: aat tct aat acg act cac tat agg gAG AGG TTC GGG CGC CAC TGC TAG A

SEQ ID 11: aat tct aat acg act cac tat agg gCGGGCGCCACTGCTA

SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT

SEQ ID 9-11: actually include the sequence as reflected in SEQ ID 1-3. In SEQ ID 9-11, the sequences of SEQ ID 1-3 are operably linked to a promoter sequence (the T7 promoter sequence). This makes the sequences especially suitable for use as an upstream primer in a transcription-based amplification technique such as NASBA.

A preferred embodiment of the present invention is that which consists of a combination of two oligonucleotides according to the invention, for use as a group in nucleotide acid amplification.

Said pair of oligonucleotides, for use as a group in the amplification of a target sequence located within the LTR region of the HIV-1 genome, consists of a first oligonucleotide of 10-50 nucleotides in length and comprising at least one fragment of 10 nucleotides, of a sequence selected from the group consisting of:

SEQ ID 1: G GGC GCC ACT GCT AGA GA SEQ ID 2: G TTC GGG CGC CAC TGC TAG A

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SEQ ID 3: CGGGCGCCACTGCTA and a second oligonucleotide 10-50 nucleotides in length and comprising at least one 10 nucleotide fragment of a sequence selected from the group consisting of:

SEQ ID 4: CTG CTT AAA GCC TCA ATA AA

SEQ ID 5: CTC AAT AAA GCT TGC CTT GA

SEQ ID 12: GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT

One of the oligonucleotides can serve as an "upstream oligonucleotide", ie an upstream primer, while the second oligonucleotide serves as a "downstream oligonucleotide", ie, a downstream primer, in the amplification reaction. The location in the HIV genome (or the complementary sequence thereof) with which the two oligonucleotides comprised in said pair according to the invention can be associated, will jointly define the sequence of the nucleic acid to be amplified. The amplified sequence is located between the "primer binding sites" within the LTR region of the HIV genome. It has been found that, by using an oligonucleotide pair according to the invention in the amplification reaction, a precise and reliable amplification of nucleic acid derived from all currently known HIV subtypes can be achieved.

A particularly preferable pair of oligonucleotides according to the invention will consist of a first primer comprising SEQ ID NO 1 and a second primer with the sequence SEQ ID NO 5. For use in a transcription-based amplification method, the oligonucleotide with SEQ ID NO 9, in combination with an oligonucleotide with the sequence of SEQ ID NO 5.

Part of the oligonucleotides according to the invention are particularly suitable for use as a probe in the detection of nucleic acid amplified with a pair of oligonucleotides according to the invention. When used as a probe, a detectable tag can be incorporated into such oligonucleotides. The oligonucleotides according to the invention which are particularly suitable as probes consist essentially of the sequence:

SEQ ID 6: TCT GGT AAC TAG AGA TCC CTC

SEQ ID 7: TAG TGT GTG CCC GTC TGT or

SEQ ID 8: AGT GTG TGC CCG TCT GTT equipped with a detectable tag. A particularly preferable oligonucleotide in this sense is an oligonucleotide with a sequence as represented in SEQ ID 6.

Several labeling fractions are known in the art. Said fraction can be for example a radioactive compound, a detectable enzyme (eg horseradish peroxidase (HRP)), a hapten such as biotin, or any other fraction capable of generating a detectable signal such as, for example, colorimetric, fluorescent, chemiluminescent or electrochemiluminescent. Hybrids between oligonucleotides according to the invention and target nucleic acid (amplified) can also be detected by other methods known to those skilled in the art. Obviously, the methods for nucleic acid amplification, like those mentioned above, by using the oligonucleotides according to the present invention are also part of the present invention.

The present invention also provides assay kits for the amplification and detection of HIV nucleic acid. The use of these test kits allows for the accurate and sensitive screening of samples presumed to contain HIV-derived nucleic acid. Said test kits can contain a pair of oligonucleotides according to the invention and, optionally, also an oligonucleotide according to the invention that can be used as a probe for the detection of the amplified material. Furthermore, the test kit may contain suitable amplification reagents. Such reagents are, for example, the enzymes suitable for carrying out the amplification reaction. A kit, adapted for use with NASBA, for example, may contain suitable amounts of reverse transcriptase, RNase H, and T7 RNA polymerase. Said enzymes can be present in the kit in a buffered solution, although they can also be provided as a lyophilized composition, for example, lyophilized spherical particles. Said lyophilized particles have been previously described in PCT application No. EP95 / 01268. The kit can also be equipped with buffer compositions, suitable for carrying out an amplification reaction. Such buffers can be optimized for the particular amplification technique for which the kit is intended, as well as for use with the particular oligonucleotides that are provided in the kit. In transcription-based amplification techniques, such as NASBA, such buffers may contain, for example, DMSO, which enhances the amplification reaction (as described in PCT application No. US 90/04733).

Furthermore, an internal control can be included in the kit as a means of checking the amplification procedure and to prevent false negative test results from occurring as a result of failures in the amplification procedure. The use of internal controls in amplification techniques based on

ES 2 153 807 T3 in transcription is described in PCT application No. EP 93/02248. The optimal control sequence is selected so that it does not compete with the target nucleic acid in the amplification reaction. Kits may also contain reagents for the isolation of nucleic acid from a biological specimen prior to amplification. A suitable method for nucleic acid isolation is described in EP 389063.

Brief description of the figures

Figure 1

Products of amplification after staining and detection by enhanced chemiluminescence obtained after amplification in the presence (panel A) or absence (panel B) of HIV-1 RNA using the following primer pairs. Lane 1: SEQ ID 9-SEQ ID 10, lane 2: SEQ ID 9-SEQ ID 5, lane 3: SEQ ID 10-SEQ ID 4, lane 4: SEQ ID 10-SEQ ID 5, lane 5: SEQ ID 3 -SEQ ID 12.

Examples

Example 1

Amplification and detection of HIV-1 genomic RNA

The following procedures were applied to amplify and detect HIV-1 genomic RNA from samples, as described in the following examples.

Sample preparation and nucleic acid isolation

Nucleic acid isolation was carried out according to Boom et al., 1990, Journal of Clinical Microbiology 28, 495-503 and European Patent No. EP 0389063. Briefly: A 200 µl volume of pooled plasma of healthy donors (negative for HBsAg, anti-HIV and anti HCV) to 900 μl of lysis buffer (47 mM Tris-HCl, pH 7.2, 20 mM EDTA, 1.2% Trition X-100, guanidine thiocyanate 4.7 M (GusSCN, Fluka, Buchs, Switzerland). After vortexing and centrifugation (30 seconds, 13,000 rpm) a series of standard HIV-1 subtype B RNA dilutions were added, characterized and described by Layne et al., 1992, Virology 189, 695-714 or specimens HIV-1 positive. After the addition of 50 µl of activated silicon (0.4 g / ml suspension in 0.1 N HCl), the suspension was incubated for 10 minutes at room temperature with regular vortexing. After centrifugation (3060 seconds, 13,000 rpm), the silicon conglomerate was washed twice with 1 ml of wash buffer (5.25 M GuSCN, 50 mM Tris-HCl, pH 6.4), followed by two washes with 70% ethanol and an acetone wash step. The silicon conglomerate was then dried for ten minutes in a heating block at 56 ° C. Nucleic acids were eluted on silicon by adding 50 µl of elution buffer (1.0 mM Tris-HCl, pH 8.5) and incubated at 56 ° C for 10 minutes. Next, 5 µl of the conglomerate eluate was taken for later use in the amplification reaction. The remainder of the eluate was stored at -70 ° C.

NASBA amplification

Amplifications were carried out in a 20 µl reaction volume comprised of 10 µl of primer mix, 5 µl (isolate) of nucleic acids and 5 µl of enzyme mix. The primer mix was obtained by reconstitution of a lyophilized accusphere in 50 µl of accusphere diluent, 51.6 µl of water, 8.4 µl of 2M KCl and 5 µl of each primer (10 juM). The mixture was then thoroughly vortexed prior to use. From this first primer mix, 10 µl to 5 µl of HIV-1 RNA (isolated) (standard) was added. The mixture was incubated for 5 minutes at 65 ° C and then incubated at 41 ° C for another 5 minutes. After these incubations, 5 µl of the enzyme mixture was added and incubated for 5 minutes at 41 ° C. The tubes were transferred to the detection zone and incubated for 90 minutes at 41 ° C. After the amplification reaction, the tubes were stored at -20 ° C until later use.

The amplification reaction contained the following reagents:

mM Tris-HCl, pH 8.5 mM MgCl₂ mM KCl

15% v / v mM dimethyl sulfoxide mM dithiothreitol of each mM deoxynucleoside triphosphate of the nucleosides rATP, rCTP, rUTP 1.5mMrGTP

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0.5 mM ITP

0.105 pg / pl BSA

0.08 units RNaseH units, T7 RNA polymerase

6.4 units reverse transcriptase from poultry myeloblastosis virus (Seikagaku, USA) 0.2 pM of each primer

375 mM sorbitol

45.6 mM sucrose

28.5 mM hand

0.13 mM dextran T40 Detection of amplified products

A. Gel electrophoresis

The presence of amplified products was analyzed using an agarose gel (100 ml of 2% Pronarose and 0.5 pg / ml of ethidium bromide) and using 1'TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8 , 0) as a shift buffer. Electrophoresis was carried out at 100 volts for approximately 30 minutes. The ethidium bromide stained bands of the amplified products were visualized using UV radiation. The spot was hybridized with biotin probe (3 pM) in a hybridization mix (750 mM NaCl, 75 mM sodium citrate, 20 mM Na2HPO₄/ NaH₂PO₄ (pH 6.7), 10 * Denhardts) incubating the spot for 4 hours at 50 ° C. After hybridization, the spot was washed twice for 5 minutes at 50 ° C with 450 mM NaCl, 45 mM sodium citrate, pH 6.4 (2 * SSPE) and 1% sodium dodecyl sulfate solution (SDS) and a once for 10 minutes with 20 mM Na₂HPO₄, pH 7.4, Na 360 mM, EDTA 2 mM and SDS 0.1% at room temperature. The spot was then incubated for 30 minutes, with 2 μl of streptavidin / horseradish peroxidase solution (500 U / ml from Amersham Life Science Enhanced Chemiluminescence Detection Kit) in 10 ml of Na₂HPO₄ 50 mM, 900 mM NaCl, 5 mM EDTA, pH 7.4 and 0.5% SDS. After the washes, respectively twice for 5 minutes in 2 * SSPE, 0.1% SDS and once for 10 minutes in 2 * SSPE at room temperature, the stain was dried between fabrics, developed and exposed to a film according to the Amersham team protocol.

B. Electrochemiluminescence (ECL) probe hybridization

Amplification products were diluted twice in detection diluent (1.0 mM Tris / HCl, pH 8.5 and 0.2 g / l 2-methylisothiazolone HCl). Next, 5 µl of the diluted amplification product was incubated for 30 minutes at 41 ° C with 0.084 pm of the HIV-1 specific biotin probe bound to 5 µg of streptavidin-coated magnetic beads (mean size 28 pm ± 0, 2 pm, Dynal, Great Neck, NY, USA) and 2 * 10 molecules of a probe labeled with ECL complex (Tris (2,2-bipyridine] ruthenium [n], in a total volume of 25 µl, 750 mM NaCl , sodium citrate, 75 mM, pH 6.4 (5 * SSC). As negative controls, the detection diluent was also incubated with the probe-bead and the ECL-probe mixtures. During the incubation, the tubes were shaken every 10 minutes to keep the beads in suspension. Next, 300 μl of the assay buffer solution (100 mM tripropylamine, pH 7.5) was added and the tubes were placed in an ECL detection instrument (NASBA QR-system from Organon Teknika BV) for reading of the emitted ECL signals.

Example 2

Amplification and detection of HIV-1 genomic RNA

The following primer pairs were tested in 10⁴ copies (as determined by spectrophotometry at 260 nm) of the HIV-1 RNA standard. RNA was added directly in amplification. Analysis of the amplified products by gel electrophoresis was carried out as described in Example 1. The results are shown in Figure 1.

All primer and probe pairs of the present invention were capable of amplifying and detecting HIV-1 subtype B RNA to a similar degree. Analytical sensitivity for two primer pairs (SEQ ID 9 / SEQ ID 5 and SEQ ID 11 / SEQ ID 5) is shown using a dilution series of the HIV-1 RNA standard that has an initial concentration of 5.5 '10⁹ copies / ml. Detection was carried out with probes having the sequences represented in SEQ ID 7 and SEQ ID 6. Amplification and detection was carried out as described in example 1. Table 1 shows the results obtained with both pairs of primers.

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TABLE 1

<td>HIV-1 RNA copies</td><td colspan="2">SEQ ID 3-SEQ ID 5</td><td colspan="2">SEQ ID 9-SEQ ID 5</td>

<td></td><td>No. detected</td><td>% detected</td><td>No. detected</td><td>% detected</td>

<td> 200</td><td> 4:4</td><td> 100 %</td><td> 4:4</td><td> 100%</td>

<td> 100</td><td> 8:8</td><td> 100%</td><td> 8:8</td><td> 100%</td>

<td> 50</td><td> 8:8</td><td> 100%</td><td> 8:8</td><td> 100%</td>

<td> 25</td><td> 7:8</td><td> 87,5 %</td><td> 7:8</td><td> 87,5 %</td>

<td> 12</td><td> 6:8</td><td> 75%</td><td> 4:8</td><td> 50%</td>

<td> 6</td><td> 2:8</td><td> 25%</td><td> 4:7</td><td> 57%</td>

<td> 3</td><td> 0:8</td><td> 0%</td><td> 2:7</td><td> 28,5 %</td>

<td> 0</td><td> 0:8</td><td> 0%</td><td> 0:7</td><td> 0%</td>

Both primer pairs have approximately the same sensitivity for the HIV-1 RNA standard, respectively, a detection rate of 70% for the primer pair SEQ ID 9 / SEQ ID 5 and a detection rate of 63% for the pair. primer SEQ ID 3 / SEQ ID 5.

Example 3

Amplification and detection of HIV-1 RNA in the presence of an internal control

In this example, the primer pair SEQ ID 9 / SEQ ID 5 and a probe with the sequence SEQ ID 7 were tested in a dilution series of the HIV-1 RNA standard in the presence of an internal control (ic-) RNA. The ic-RNA is an in vitro transcript that contains part of the LTR sequence of HIV-1 HXB-2 and is added10⁴ copies (as determined by spectrophotometry at 260 nm) prior to nucleic acid isolation. Isolation, amplification and ECL detection were carried out as described in Example 1. For comparative purposes, separate amplification and detection was carried out using a primer pair located in the gag region of HIV- 1 (P1 / P2) previously described by Van Gemen et al., 1993, Journal of Virological Methods 43, 177-188. The probes used to detect the amplification products generated by the gag-based primer pair were:

Biotin probe: 5'-TGTTAAAAGAGACCATCAATGAGGA

ECL probe: 5'-GAATGGGATAGAGTGCATCCAGTG

Table 2 shows the results.

TABLE 2

<td>HIV-1 RNA Copies / 200μΙ</td><td colspan="2">SEQ ID 9-SEQ ID 5 LTR</td><td colspan="2">GAG P1 / P2</td>

<td></td><td>No. detected</td><td>% detected</td><td>No. detected</td><td>% detected</td>

<td> 320</td><td> 9:10</td><td> 90 %</td><td>ne *</td><td>ne *</td>

<td> 160</td><td> 5:10</td><td> 50 %</td><td> 8:10</td><td> 80%</td>

<td> 80</td><td> 5:10</td><td> 50%</td><td> 5:10</td><td> 50%</td>

<td> 40</td><td> 3:8</td><td> 38 %</td><td> 4:10</td><td> 40%</td>

<td> 20</td><td> 3:10</td><td> 32 %</td><td> 1:10</td><td> 10%</td>

<td> 10</td><td> 1:10</td><td> 10 %</td><td> 2:10</td><td> 20%</td>

<td> 0</td><td> 0:5</td><td> 0 %</td><td> 1:5</td><td> 20%</td>

ne means not tested

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Both the LTR primer pair in this example and the gag primer pair were able to detect a similar amount of HIV-1 RNA in an in vitro produced RNA-controlled assay that was added prior to nucleic acid isolation.

Example 4

Detection of HIV-1 RNA in HIV-1 positive samples

In this example, 40 HIV-1 positive samples from various geographic locations were tested for the presence of HIV-1 RNA. All samples were isolated through the nucleic acid isolation method described in example 1. Amplification and detection was carried out as described in example 1 using a primer pair SEQ ID 9 / SEQ ID 5 and probe with the sequence SEQ ID 7 or probe with the sequence of SEQ ID 8.

For comparative purposes, separate amplification and detection was carried out using the gag primer pair and probes as described in Example 3. The presence of amplified products was detected by ECL probe hybridization according to the method of Example 1 Table 3 shows the results.

TABLE 3

<td>Primer torque</td><td>ECL probe</td><td>No. detected</td><td>% detected</td>

<td>SEQ ID 9 / SEQ ID 5 (LTR)</td><td>SEQ ID 7</td><td> 40/40</td><td> 100%</td>

<td>SEQ ID 9 / SEQ ID 5 (LTR)</td><td>SEQ ID 8</td><td> 10/10</td><td> 100%</td>

<td>P1 / P2 (gag)</td><td></td><td> 29/40</td><td> 72,5 %</td>

The two probe-primer combinations derived from the HIV-1 LTR region described in this example were capable of detecting HIV-1 from all samples tested. In contrast, the gag assay primer probe combination did not detect HIV-1 genomic RNA from a large number of samples.

Example 5

Amplification and detection of HIV-1 RNA with defined subtypes

In this example, 33 samples containing HIV-1 RNA of known envelope-type subtypes were tested. The samples originate from subtyped viruses propagated in cell culture. Cell culture supernatant samples of both variants of group M subtypes from A to H and group O variants were spiked into HIV-1 negative human plasma and treated according to the method of Example 1. Amplification and detection was carried out essentially as in Example 4. Table 4 shows the results as a number of samples of each type from which HIV-1 RNA was detected out of the total number of each type tested.

TABLE 4

<td></td><td colspan="10">HIV-1 types</td>

<td>Primer torque</td><td>TO</td><td>B</td><td>C</td><td>D</td><td>AND</td><td>F</td><td>G</td><td>H</td><td>OR</td><td>Total</td>

<td>SEQ ID 9 / SEQ ID 5 (LTR)</td><td> 5:5</td><td> 2:2</td><td> 2:2</td><td> 5:5</td><td> 7:7</td><td> 1:1</td><td> 1:1</td><td> 1:1</td><td> 9:9</td><td> 33:33</td>

<td></td><td> 4:5</td><td> 2:2</td><td> 2:2</td><td> 5:5</td><td> 6:7</td><td> 1:1</td><td> 1:1</td><td> 1:1</td><td> 0:9</td><td> 22:33</td>

HIV-1 RNA was detected from the 33 HIV-1 samples using the LTR primer pair SEQ ID 9 / SEQ ID 5 and the probe with the sequence SEQ ID 7. In contrast, the assay in which the combination of primer probes was used gag as described in Example 3 failed to detect subtype A and subtype E from each of the samples and all samples containing HIV-1 RNA from group O members.

IS 2 153 807 T3

Example 6

Amplification and detection of clinical samples of HIV-1

In this example, 7 samples obtained from seropositive patients of African, South American, and Asian origin were tested for the presence of HIV-1 genomic RNA. Despite the fact that blood samples from these patients are positive for anti-p24 antibody (Abbott Laboratories, Abbott Park, IL) and Western blot (Genelabs), All negative specimens were scored using the QL HIV-1 NASBA RNA assay (Organon Teknika BV) using the gag primer pair probe combination from Example 3 and only a single sample (R9612222) was detected and quantitatively determined ( 29,500 RNA copies / ml) by the Quantiplex HIV-1 RNA 2.0 Assay (Chiron) using a sample volume of 50 µl. In contrast, using an equal sample volume, the primer pair SEQ ID 9 / SEQ ID 5 and the probe with the sequence SEQ ID 7 detected four out of seven samples (Table 5).

TABLE 5

<td>Sample code</td><td>Country</td><td>SEQ ID 9 / SEQ ID 5 (LTR)</td>

<td>R9610155</td><td>Thailand</td><td>positive</td>

<td>R9612222</td><td>Ghana</td><td>positive</td>

<td>R9600062</td><td>Brazil</td><td>negative</td>

<td>R9612218</td><td>Zaire</td><td>negative</td>

<td>R9611710</td><td>Liberia</td><td>positive</td>

<td>R9610718</td><td>Antilles</td><td>positive</td>

<td>R9607884</td><td>Rwanda</td><td>negative</td>

Samples lost by the LTR primer probe combination are not detected due to a low viral load below the detection level, as using a 1 ml sample volume, the Quantiplex HIV-1 RNA 2.0 Assay (Chiron ) quantitatively determined HIV-1 RNA levels of 30 or below 30 copies of HIV-1 RNA per 50 µl of these samples.