Cross-reactive primers for amplifying the nucleic acids of HIV-1 and HIV-2
View Patent ↗Compositions, methods and kits for detecting the nucleic acids of HIV-1, HIV-2, or the combination of HIV-1 and HIV-2. Particularly described are oligonucleotides that are useful as hybridization probes and amplification primers, including cross-reacting hybridization probes and cross-reacting amplification primers, for detecting very low levels of viral nucleic acids.
1. A composition for amplifying both human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) nucleic acids that may be present in a biological sample, comprising:
(a) a first primer consisting of SEQ ID NO:59 and optionally, attached at the 5′ end thereof, a first primer upstream sequence that comprises a restriction endonuclease cleavage site or an RNA polymerase promoter; and
(b) a second primer consisting of SEQ ID NO:61 and optionally, attached at the 5′ end thereof, a second primer upstream sequence that comprises a restriction endonuclease cleavage site or an RNA polymerase promoter.
2. The composition of claim 1 , wherein said composition does not comprise a pair of HIV-2-specific primers for amplifying only said HIV-2 nucleic acid and not said HIV-1 nucleic acid.
3. The composition of claim 2 , wherein said composition further comprises a pair of HIV-1-specific primers for amplifying only said HIV-1 nucleic acid and not said HIV-2 nucleic acid.
4. The composition of claim 1 , wherein said composition further comprises a pair of HIV-1-specific primers for amplifying only said HIV-1 nucleic acid and not said HIV-2 nucleic acid.
5. The composition of claim 1 , wherein said first primer and said second primer are each up to 60 bases in length.
6. The composition of claim 5 , where said first primer consists of SEQ ID NO:53 and optionally, attached at the 5′ end thereof, said first primer upstream sequence.
7. The composition of claim 5 , where said first primer consists of SEQ ID NO:54 and optionally, attached at the 5′ end thereof, said first primer upstream sequence.
8. The composition of claim 5 , wherein the second primer is not attached to said second primer upstream sequence.
9. The composition of claim 8 , wherein said second primer is selected from the group consisting of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.
10. The composition of claim 5 , wherein the first primer is not attached to said first primer upstream sequence.
11. The composition of claim 10 , wherein the second primer is not attached to said second primer upstream sequence.
12. A method of determining whether a test sample contains at least one of HIV-1 nucleic acid and HIV-2 nucleic acid, said method comprising the steps of:
(a) combining said test sample with a composition according to claim 1 to form a reaction mixture;
(b) amplifying, in an in vitro nucleic acid amplification reaction, nucleic acid present in said reaction mixture of step (a); and
(c) detecting an HIV-1 amplicon, and HIV-2 amplicon, or a combination of both amplicons synthesized in step (b), thereby determining that said test sample contains at least one of HIV-1 nucleic acid and HIV-2 nucleic acid.
13. The method of claim 12 , wherein the detecting step comprises detecting in a hybridization reaction.
14. The method of claim 13 , wherein said hybridization reaction in the detecting step comprises a cross-reactive probe which hybridizes independently to said HIV-1 amplicon and to said HIV-2 amplicon.
15. The method of claim 14 , wherein said hybridization reaction in the detecting step further comprises an HIV-1 specific probe which hybridizes only to said HIV-1 amplicon and not to said HIV-2 amplicon.
16. The method of claim 12 , wherein said in vitro nucleic acid amplification reaction in the amplifying step is a multiplex amplification reaction that can further amplify, with an assortment of target-specific primers, at least one nucleic acid which is different from HIV-1 and HIV-2.
17. The method of claim 16 , wherein said at least one nucleic acid which is different from HIV-1 and HIV-2 is selected from the group consisting of HBV and HCV.