Kit for detecting HIV-1 and method for detecting HIV-1 using the same
View Patent ↗A method is described for the real-time detection of HIV-1 gene target DNA in a sample, including obtaining HIV-1 specific cDNA by reverse transcription, amplifying a portion of the cDNA, and detecting the amplicons so produced using probe labeled with a detectable marker.
1. A kit for detecting HIV-1, comprising:
a first primer comprising the oligonucleotide of the nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8:
(SEQ ID NO: 1)
GCAGTACAAATGGCAGTATTCATCCACAATT,
(SEQ ID NO: 2)
CAGCAGTACAAATGGCAGTATTCATCCA,
(SEQ ID NO: 3)
CAGGAATTCGGGATACCCTACAATCCTCAA,
(SEQ ID NO: 4)
AGGAATTCGGGATACCCTACAATCCTCAAA,
(SEQ ID NO: 5)
ACAATCCTCAAAGTCAGGGAGTAGTAGAAT,
(SEQ ID NO: 6)
CAAAGTCAGGGAGTAGTAGAATCCATGAAT,
(SEQ ID NO: 7)
CCAAGGGGAAGTGACATAGCAGGAACTACTA,
and
(SEQ ID NO: 8)
CCAAGGGGAAGTGACATAGCAGGAACTACT.
a second primer comprising the oligonucleotide of the nucleotide sequence selected from the group consisting of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19:
(SEQ ID NO: 9)
CTGACAGGGCTATACATTCTTACTATTTTATT,
(SEQ ID NO: 10)
TTTCTGCTGTCCCTGTAATAAACCCGAAAATTT,
(SEQ ID NO: 11)
TCTCTGCTGTCCCTGTAATAAACCCGAAAATTT,
(SEQ ID NO: 12)
CTCTGCTGTCCCTGTAATAAACCCGAAAATTT,
(SEQ ID NO: 13)
TCTGCTGTCCCTGTAATAAACCCGAAAATTT,
(SEQ ID NO: 14)
CTCTGCTGTCTCTGTAATAGACCCGAAAATTT,
(SEQ ID NO: 15)
CTCTGCTGTCTCTGTAATAGACCCGAAAATT,
(SEQ ID NO: 16)
CTCTGCTGTCTCTGTAATAGACCCGAAAATTTT,
(SEQ ID NO: 17)
GTCCTTTCCAAATAGGATCTCTGCTATCTC,
(SEQ ID NO: 18)
CCAAATAGGATCTCTGCTATCTCTGTAATA,
and
(SEQ ID NO: 19)
AAATAGGATCTCTGCTATCTCTGTAATAGA,
and
a probe comprising the oligonucleotide of the nucleotide sequence selected from the group consisting of SEQ ID NO: 20, 21, 22, 23, 24 and 25:
(SEQ ID NO: 20)
TACCCTTCAGrGrArArCAAATAGGATGGAT,
(SEQ ID NO: 21)
GGAGAAATTTATAArArArGrATGGATAATCCTG,
(SEQ ID NO: 22)
TTAAAAGAAAAGGGGrGrGrArUTGGGGGGTACA,
(SEQ ID NO: 23)
TTAAAAGAAAAGGGGGrGrArUrUGGGGGGTACA,
(SEQ ID NO: 24)
TGTACCCCCCArArUrCrCCCCCTTTTCTTTTAA,
and
(SEQ ID NO: 25)
TTAAAAGAAAAGG*GG*rGrGrArUTG*GG*GG*GTACA,
wherein the nucleotides “rA,” “rG,” “rU, and “rC” are ribonucleotides and the nucleotide G* is 8-Aza-7-Deaza-dG.
2. The kit of claim 1 , which further comprises an amplifying polymerase activity and an RNase H activity.
3. The kit of claim 1 , which further comprises a reverse transcriptase activity.
4. The kit of claim 1 , wherein a 5′ end of each probe is labeled with a fluorescence label or a 3′ end of each of the probes is labeled with a fluorescence quencher.
5. The kit of claim 1 , further comprising a mixture comprising dATP, dCTP, dGTP, and dTTP; a DNA polymerase; RNase HII; and a buffer solution.
6. The kit of claim 1 , further comprising uracil-N-glycosylase.
7. The kit of claim 1 , wherein the probe is linked to a solid support.
8. The kit of claim 1 , wherein the probe is present as a free form in a solution.
9. The kit of claim 2 , wherein the amplifying polymerase activity is the activity of a thermostable DNA polymerase.
10. The kit of claim 2 , wherein the RNase H activity is the activity of a thermostable RNase H.
11. The kit of claim 2 , wherein the RNase H activity is a hot start RNase H activity.