ASSAY FOR DETECTING AND QUANTIFYING HIV-1
Method of detecting HIV-1 nucleic acids using nucleic acid amplification and a molecular torch hybridization probe. The invented method is characterized by high levels of precision in the quantitation of HIV-1 targets at low copy numbers, and by accurate detection of different HIV-1 subtypes, including M group and O group variants.
1 . A method of quantifying HIV-1 M group nucleic acids and HIV-1 O group nucleic acids in a test sample that comprises nucleic acids, said method comprising the steps of:
(a) contacting nucleic acids of the test sample with a first amplification primer, a second amplification primer, and a molecular torch hybridization probe, the base sequence of the molecular torch hybridization probe being CGGIGGGUACAGUGCCCCCG (SEQ ID NO:24);
(b) amplifying, in an isothermal in vitro nucleic acid amplification reaction, any HIV-1 M group and HIV-1 O group nucleic acids that may be present in the test sample using said first and second amplification primers;
(c) monitoring, with the molecular torch hybridization probe, the time-dependent production of amplification products in the isothermal in vitro nucleic acid amplification reaction to determine a time-dependent value indicative of the combined starting quantity of HIV-1 M group and HIV-1 O group nucleic acids present in the isothermal in vitro nucleic acid amplification reaction; and
(d) quantifying the combined amounts of HIV-1 M group and HIV-1 O group nucleic acids present in the test sample using a standard curve and the time-dependent value determined in step (c).
2 . The method of claim 1 ,
wherein the standard curve used in step (d) relates pre-amplification amounts of an HIV-1 standard polynucleotide, and the time required to achieve a threshold level of amplification for the HIV-1 standard polynucleotide in the isothermal in vitro nucleic acid amplification reaction, and
wherein the difference between the number of starting copies per reaction quantified in step (d) and the actual number of starting copies per reaction is no greater than 0.5 log 10 copies per reaction for independent isothermal in vitro amplification reactions conducted using HIV-1 M group subtype-B nucleic acid templates, and HIV-1 O group templates, each at actual starting levels of 1,000 copies per reaction.
3 . The method of claim 2 ,
wherein the HIV-1 standard polynucleotide is an HIV-1 subtype B nucleic acid.
4 . The method of claim 1 ,
wherein the first amplification primer in step (a) is a promoter-primer that consists of a phage promoter sequence joined upstream of SEQ ID NO:15,
wherein the isothermal in vitro nucleic acid amplification reaction in step (b) is a transcription mediated amplification reaction that synthesizes RNA amplification products, and
wherein step (c) comprises monitoring, with the molecular torch hybridization probe, the time-dependent production of RNA amplification products in the transcription mediated amplification reaction.
5 . The method of claim 4 ,
wherein the standard curve used in step (d) relates pre-amplification amounts of an HIV-1 standard polynucleotide, and the time required to achieve a threshold level of amplification for the HIV-1 standard polynucleotide in the isothermal in vitro nucleic acid amplification reaction, and
wherein the HIV-1 standard polynucleotide comprises either an HIV-1 M group standard or an HIV-1 O group standard.
6 . The method of claim 4 ,
wherein the second amplification primer in step (a) is selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
7 . The method of claim 4 ,
wherein the second amplification primer in step (a) is SEQ ID NO:2.
8 . The method of claim 4 ,
wherein the second amplification primer in step (a) is SEQ ID NO:5.
9 . The method of claim 8 ,
wherein the standard curve used in step (d) relates pre-amplification amounts of an HIV-1 standard polynucleotide, and the time required to achieve a threshold level of amplification for the HIV-1 standard polynucleotide in the isothermal in vitro nucleic acid amplification reaction, and
wherein the HIV-1 standard polynucleotide is an HIV-1 M group standard, and not an HIV-1 O group standard.
10 . The method of claim 9 ,
wherein the difference between the number of starting copies per reaction quantified in step (d) and the actual number of starting copies per reaction is no greater than 0.5 log 10 copies per reaction for independent isothermal in vitro amplification reactions conducted using HIV-1 M group subtype-B nucleic acid templates, and HIV-1 O group templates, each at actual starting levels of 1,000 copies per reaction.
11 . The method of claim 9 ,
wherein the HIV-1 M group standard is an HIV-1 subtype B nucleic acid.
12 . The method of claim 1 ,
wherein the standard curve used in step (d) relates pre-amplification amounts of an HIV-1 standard polynucleotide, and the time required to achieve a threshold level of amplification for the HIV-1 standard polynucleotide in the isothermal in vitro nucleic acid amplification reaction, and
wherein the HIV-1 standard polynucleotide in step (d) comprises either an HIV-1 M group standard or an HIV-1 O group standard.
13 . The method of claim 12 ,
wherein the HIV-1 M group standard is an HIV-1 subtype B nucleic acid.
14 . The method of claim 13 ,
wherein the difference between the number of starting copies per reaction quantified in step (d) and the actual number of starting copies per reaction is no greater than 0.5 log 10 copies per reaction for independent isothermal in vitro amplification reactions conducted using HIV-1 M group subtype-B nucleic acid templates, and HIV-1 O group templates, each at actual starting levels of 1,000 copies per reaction.
15 . The method of claim 14 ,
wherein the first amplification primer in step (a) is a promoter-primer that consists of a phage promoter sequence joined upstream of SEQ ID NO:15,
wherein the isothermal in vitro nucleic acid amplification reaction in step (b) is a transcription mediated amplification reaction that synthesizes RNA amplification products, and
wherein step (c) comprises monitoring, with the molecular torch hybridization probe, the time-dependent production of RNA amplification products in the transcription mediated amplification reaction.
16 . The method of claim 1 ,
wherein the molecular torch hybridization probe of step (a) comprises a fluorophore moiety and a quencher moiety, and
wherein step (c) comprises monitoring a fluorescent signal generated by the fluorophore moiety.
17 . The method of claim 16 ,
wherein nucleotide positions 15 and 16 of the molecular torch hybridization probe are joined by a non-nucleotide linker, and
wherein a fluorescein moiety is joined at one terminus of SEQ ID NO:24.
18 . The method of claim 1 ,
wherein said standard curve is stored electronically in a memory device of a testing instrument.
19 . The method of claim 1 ,
wherein the difference between the number of starting copies per reaction quantified in step (d) and the actual number of starting copies per reaction is no greater than 0.5 log 10 copies per reaction for independent isothermal in vitro amplification reactions conducted using HIV-1 M group subtype-B nucleic acid templates, and HIV-1 O group templates, each at actual starting levels of 1,000 copies per reaction.
20 . The method of claim 1 ,
wherein nucleotide positions 15 and 16 of the molecular torch hybridization probe are joined by a non-nucleotide linker, and
wherein a fluorescein moiety is joined at one terminus of SEQ ID NO:24.