HIV type and subtype detection
The present invention relates to the detection of HIV by amplification and PCR-based methods.
1. A polynucleotide selected from the group consisting of:
SEQ ID NO: 2 and the fragments of SEQ ID NO: 2 selected from SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308;
SEQ ID NO: 4 and the fragments of SEQ ID NO: 4 selected from SEQ ID NO: 310, SEQ ID NO: 311;
SEQ ID NO: 5
SEQ ID NO: 1 and the fragment thereof of SEQ ID NO: 305; and
SEQ ID NO: 3 and the fragment thereof of SEQ ID NO: 309.
2. An amplicon obtainable by implementation of a process for the detection of at least one HIV target on a HIV-containing sample, wherein said process comprises the step of producing at least one amplicon by means of at least two oligonucleotides, wherein said at least two oligonucleotides are primers which are structurally suitable for specifically amplifying at least one reference template sequence selected from the group consisting of:
SEQ ID NO: 2 and the fragments of SEQ ID NO: 2 selected from SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308;
SEQ ID NO: 4 and the fragments of SEQ ID NO: 4 selected from SEQ ID NO: 310, SEQ ID NO: 311;
SEQ ID NO: 5;
SEQ ID NO: 1 and the fragment thereof of SEQ ID NO: 305; and
SEQ ID NO: 3 and the fragment thereof of SEQ ID NO: 309;
wherein said primers are structurally suitable for annealing to said HIV reference isolate at such positions that they target on said HIV reference isolate a sequence, which consists of said reference template sequence,
wherein the nucleotide length of said amplicon is the nucleotide length of a polynucleotide of claim 1 .
3. An amplification composition comprising at least one amplicon of claim 2 .
4. The amplicon of claim 2 , wherein said process further comprises the step of detecting said amplicon by means of at least one probe.
5. The amplicon of claim 4 , wherein each of the 5′ and 3′ ends of said at least one probe is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said at least one probe when said at least one probe is not hybridized to said amplicon.
6. An isolated oligonucleotide suitable for HIV detection, selected from the group consisting of:
SEQ ID NOs: 24, 190, 221, 276, 297 and 300, and
SEQ ID NOs: 190, 297 and 300, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 190, 297 or 300 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 190, 297 or 300 when said sequence of SEQ ID NO: 190, 297 or 300 is unhybridized.
7. The isolated oligonucleotide of claim 6 consisting of
SEQ ID NO: 24.
8. The isolated oligonucleotide of claim 6 , selected from the group consisting of:
SEQ ID NO: 190; and
SEQ ID NO: 190, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 190 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 190 when said sequence of SEQ ID NO: 190 is unhybridized.
9. The isolated oligonucleotide of claim 6 consisting of
SEQ ID NO: 221.
10. The isolated oligonucleotide of claim 6 consisting of
SEQ ID NO: 276.
11. The isolated oligonucleotide of claim 6 , wherein said oligonucleotide is fluorescently labelled.
12. A set of oligonucleotides, comprising at least two oligonucleotides selected from a first group consisting of:
the oligonucleotide consisting of SEQ ID NO: 12,
the oligonucleotide consisting of SEQ ID NO: 24, and
an oligonucleotide selected from a second group consisting of: SEQ ID NO: 118, and
SEQ ID NO: 118, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 118 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 118 when said sequence of SEQ ID NO: 118 is unhybridized,
wherein said set specifically detects HIV.
13. A set of oligonucleotides, comprising at least one oligonucleotide selected from the group consisting of:
SEQ ID NO: 190 and
SEQ ID NO: 190, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 190 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 190 when said sequence of SEQ ID NO: 190 is unhybridized,
wherein said set specifically detects HIV.
14. A set of oligonucleotides, comprising at least one oligonucleotide selected from the group consisting of:
SEQ ID NO: 221;
SEQ ID NO: 276;
SEQ ID NO: 297; and
SEQ ID NO: 297, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 297 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 297 when said sequence of SEQ ID NO: 297 is unhybridized,
wherein said set specifically detects HIV.
15. A set of oligonucleotides, comprising at least one oligonucleotide selected from the group consisting of:
SEQ ID NO: 221;
SEQ ID NO: 276;
SEQ ID NO: 300; and
the oligonucleotide of SEQ ID NO: 300, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 300 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 300 when said sequence of SEQ ID NO: 300 is unhybridized,
wherein said set specifically detects HIV.
16. A set of oligonucleotides, comprising at least one oligonucleotide selected from the group consisting of:
SEQ ID NO: 190;
SEQ ID NO: 297;
SEQ ID NO: 300; and
SEQ ID NO: 190, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 190 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 190 when said sequence of SEQ ID NO: 190 is unhybridized;
SEQ ID NO: 297, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 297 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 297 when said sequence of SEQ ID NO: 297 is unhybridized; and
SEQ ID NO: 300, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 300 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 300 when said sequence of SEQ ID NO: 300 is unhybridized;
wherein said set specifically detects HIV.
17. A set of oligonucleotides according to any one of claims 12 - 16 , wherein at least one of said oligonucleotides is fluorescently labelled.
18. An amplicon obtainable by amplification from a HIV-containing sample with a pair of primers selected from:
a primer of SEQ ID NO: 12 and a primer of SEQ ID NO: 24; or
a primer of SEQ ID NO: 221 and a primer of SEQ ID NO: 276.
19. An amplicon, which has a nucleotide length identical to the nucleotide length of a polynucleotide of claim 1 , and which comprises a sequence having a percentage of nucleotide identity of at least 90% with a probe sequence, over the entire length of this probe sequence, wherein this probe sequence is selected from the group consisting of SEQ ID NO: 118, 190, 297, 300 and the sequences, which are complementary to these probe SEQ ID NO: sequences over the entire length of these probe SEQ ID NO: sequences.
20. An amplification composition comprising the amplicon of claim 18 or 19 .
21. A kit comprising:
a. at least one oligonucleotide selected from the group consisting of SEQ ID NOs: 24, 190, 221, 276, 297 and 300; and/or
b. at least one set of oligonucleotides of any one of claims 12 - 16 , wherein at least one of the oligonucleotides of said at least one set is optionally fluorescently labelled; and
c. optionally, instructions for the use thereof,
wherein said kit is for HIV diagnostics.
22. The isolated oligonucleotide of claim 6 , selected from the group consisting of:
the oligonucleotides of SEQ ID NOs: 297 and 300; and
the oligonucleotides of SEQ ID NOs: 297 and 300, wherein each of the 5′ and 3′ ends of said sequence of SEQ ID NO: 297 or 300 is directly linked to an oligonucleotide of 5-10 nucleotides, said 5′ end oligonucleotide and said 3′ end oligonucleotide being hybridized together and imparting a hairpin structure to said sequence of SEQ ID NO: 297 or 300 when said sequence of SEQ ID NO: 297 or 300 is unhybridized.
23. The isolated oligonucleotide of claim 6 , wherein said oligonucleotide of SEQ ID NO: 24 is one of SEQ ID NOs: 16-23 and/or said oligonucleotide of SEQ ID NO: 221 is one of SEQ ID NOs: 195-220 and/or said oligonucleotide of SEQ ID NO: 276 is one of SEQ ID NOs: 250-275.
24. The set of oligonucleotides of claim 12 , wherein said oligonucleotide of SEQ ID NO: 12 is one of SEQ ID NOs: 10-11 and/or said oligonucleotide of SEQ ID NO: 24 is one of SEQ ID NOs: 16-23 and/or said oligonucleotide of SEQ ID NO: 118 is one of SEQ ID NOs: 110-117.
25. The set of oligonucleotides of claim 14 , wherein said oligonucleotide of SEQ ID NO: 221 is one of SEQ ID NOs: 195-220 and/or said oligonucleotide of SEQ ID NO: 276 is one of SEQ ID NOs: 250-275.
26. The set of oligonucleotides of claim 15 , wherein said oligonucleotide of SEQ ID NO: 221 is one of SEQ ID NOs: 195-220 and/or said oligonucleotide of SEQ ID NO: 276 is one of SEQ ID NOs: 250-275.