madrid2i

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[0001] Process for the detection and quantification in-vitro of HIV DNA by quantitative PCR. The invention consists of developing pairs of oligonucleotides capable of hybridizing with fragments of the gag gene sequence present in the genome of the HIV virus. These oligonucleotides permit amplification of the viral DNA by quantitative PCR. Even in the case of samples with low viral load, the invention permits detection of the HIV DNA virus by juxtaposition either of a conventional PCR with a quantitative PCR, or with a double nested quantitative PCR, using different pairs of primers in each amplification. The invention, compared with the known methods, allows determining qualitatively and quantitatively, in-vitro, the presence of HIV DNA in samples, in a rapid, reproducible manner and with high sensitivity.

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1. An in-vitro method of quantitative detection of DNA of the HIV virus that comprises a double amplification of viral DNA present in the sample using a double nested quantitative PCR carried out in such a way that a first pair of oliglonucleotide PCR primers SEQ ID NO: 1 and SEQ ID NO: 2, which bind specifically to the gag gene at positions 1260 to 1482 is used in the first amplification and a second pair of oliglonucleotide PCR primers with a melting point (Tm) different from that of the first pair of primers, which bind to the gag gene inside the fragment amplified by the first pair of primers characterized in thatthe amplification and the detection of the viral DNA are carried out in a single capillary per sample with a reaction mixture containing the two pairs of primers and the probes. 2. The method according to Claim 1, characterized in thata second pair of primers SEQ ID NO: 7 and SEQ ID NO: 8, binds specifically inside the gag gene fragment amplified by the first pair or primers between positions 1286 and 1435 of said gene. 3. The method according to any of the claims 1 or 2, characterized in thatthe sequence of nucleotide of one of the probes is represented by SEQ ID NO:5. 4. The method according to any of the claims 1 to 3, characterized in thatthe sequence of nucleotide of the second probe is represented by SEQ ID NO:6. 5. The method according to Claims 1 to 4, characterized in thatthe fist pair of primers are represented by SEQ ID NO:1 and SEQ ID NO:2, the second pair of primers are represented by SEQ ID NO:7 and SEQ ID NO:8 and the sequence of nucleotides of the probes are represented by SEQ ID NO:5 and SEQ ID NO:6. 6. The method according to anyone of the preceding claims, caracterized in thatthe probes are labelled with F.R.E.T. type fluorochromes, thereafter the number of copies of viral DNA present in the test sample is quantified by extrapolating the fluorescence obtained to a previously calibrated reference straight or curve. 7. Test kit for quantifying DNA of the HIV virus, characterized in thatit comprises a reaction mixture in a single capillary that contains: i. at least one first pair of oligonucleotides PCR primers SEQ ID NO: 1 and SEQ ID NO: 2 which bind specifically to the gag gene between positions 1260 and 1482 of said gene, ii. at least one second pair of oligonucleotides PCR primers SEQ ID NO: 7 and SEQ ID NO: 8 with a melting point (Tm) different from that of the first pair of primers and which bind specifically to the gag gene inside the fragment determined by the first pairs or primers, that uses as template the product previously amplified by said first pair of primers, and,jointly or separately in a reagent, iii. a pair of oligonucleotides probes which bind specifically to the fragment of the gag gene comprised between the second pair of primers. 8. Test kit according to claim 7, characterized in thatone of the oligonucleotide probes is represented by SEQ ID NO:5. 9. Test kit according to any of the claims 7 or 8, characterized in thatthe second oligonucleotide probe is represented by SEQ ID NO:6. 10. Test kit according to any of the claims 7 to 9, characterized in thatthe first pair of oligonucleotide primers is represented by SEQ ID NO:1 and SEQ ID NO:2, the second pair of oligonucleotide primers is represented by SEQ ID NO: 7 and SEQ ID NO:8 and the pair of oligonucleotide probes is represented by SEQ ID NO:5 and SEQ ID NO:6. 11. Test kit according to any of the claims 7 to 10, characterized bythe pair of oligonucleotide probes being labelled with FRET-type fluorochromes.

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