Compositions, methods and kits for detecting 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 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 pair of cross-reactive primers to form a reaction mixture, wherein said pair of cross-reactive primers is capable of co-amplifying HIV-1 and HIV-2 nucleic acid and comprises a first primer sequence that consists of SEQ ID NO:14, optionally comprising an upstream sequence that is not complementary to either HIV-1 or HIV-2 nucleic acid, and a second primer sequence that consists of SEQ ID NO:7, optionally comprising an upstream sequence that is not complementary to either HIV-1 or HIV-2 nucleic acid;
(b) amplifying, in an in vitro nucleic acid amplification reaction, nucleic acid present in said reaction mixture of step (a); and
(c) detecting in a hybridization reaction an HIV-1 amplicon, an 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.
2. The method of claim 1 , wherein said in vitro nucleic acid amplification reaction in the amplifying step is selected from the group consisting of a Transcription Mediated Amplification (TMA) reaction, a Nucleic Acid Sequence-Based Amplification (NASBA) reaction and a Polymerase Chain Reaction (PCR) reaction.
3. The method of claim 1 , 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.
4. The method of claim 3 , 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.
5. The method of claim 4 , wherein a positive signal indicating hybridization of the cross-reactive probe together with the absence of a positive signal indicating hybridization of the HIV-1-specific probe indicates that said test sample contains HIV-2 nucleic acid and does not contain HIV-1 nucleic acid.
6. The method of claim 3 , further comprising a step for detecting in a hybridization reaction that comprises an HIV-1-specific probe said HIV-1 amplicon without also detecting said HIV-2 amplicon, wherein said HIV-1-specific probe hybridizes only to said HIV-1 amplicon and not to said HIV-2 amplicon.
7. The method of claim 6 , wherein a positive signal indicating hybridization of the cross-reactive probe together with the absence of a positive signal indicating hybridization of the HIV-1-specific probe indicates that said test sample contains HIV-2 nucleic acid and does not contain HIV-1 nucleic acid.
8. The method of claim 3 , wherein said cross-reactive probe is labeled with a homogeneously detectable label.
9. The method of claim 8 , wherein said homogeneously detectable label is a chemiluminescent label.
10. The method of claim 9 , wherein said detecting step comprises detecting with a luminometer.
11. The method of claim 1 , wherein a positive result obtained in said detecting step does not distinguish between said HIV-1 amplicon and said HIV-2 amplicon.
12. The method of claim 1 , 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.
13. The method of claim 12 , 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.