Methods for discriminating between HIV-1 and lentiviral vectors
The present disclosure provides compositions (i.e., amplification primers and probes), methods, and kits that are particularly useful for detecting and/or quantifying nucleic acids present in a sample, such as those derived from HIV or a lentiviral vector.
1. A method of detecting an amount of a lentiviral nucleic acid and an amount of an HIV nucleic acid in a sample comprising: (a) contacting the sample with a first forward primer and a first reverse primer; (b) contacting the sample with a junction probe specific for a junction site within the 3′ LTR of the lentiviral nucleic acid, wherein the junction probe comprises a first portion which is capable of hybridizing to at least a portion of a sequence within the U3 region of the lentiviral nucleic acid 3′ LTR and a second portion which is capable of hybridizing to at least a portion of a sequence within the R region of the lentiviral nucleic acid 3′ LTR, and wherein the junction probe comprises a first detectable moiety; (c) detecting signals from the first detectable moiety; (d) contacting the sample with a second probe specific for a TATA-box sequence within a 3′ LTR of an HIV nucleic acid sequence, the second probe conjugated to a second detectable moiety; and (e) detecting signals from the second detectable moiety, wherein the detection of the amount of the lentiviral nucleic acid and the amount of the HIV nucleic acid in the sample takes place in the same reaction tube.
2. The method of claim 1 , wherein the first portion of the junction probe hybridizes to a nucleotide sequence having at least 80% identity to that of SEQ ID NO: 12.
3. The method of claim 1 , wherein the second portion of the junction probe hybridizes to a nucleotide sequence having SEQ ID NO: 13.
4. The method of claim 1 , wherein the junction probe comprises a nucleotide sequence having at least 90% identity to that of SEQ ID NO: 14.
5. The method of claim 1 , wherein the second probe has a nucleotide sequence having at least 90% identity to that of SEQ ID NO: 10.
6. The method of claim 1 , wherein the first forward primer is a NuAf primer and the first reverse primer is a LTR-rev primer.
7. The method of claim 1 , further comprising detecting an amount of an HIV nucleic acid in the sample, wherein the detection of the amount of the lentiviral nucleic acid and the amount of HIV nucleic acid in the sample takes place in different reaction tubes, and wherein the detection of the amount of the HIV nucleic acid comprises contacting the sample with a second forward primer, a second reverse primer, and a second probe having a nucleotide sequence having at least 90% identity to that of SEQ ID NO: 8, wherein the second probe comprises a second detectable moiety; and detecting signals from the second detectable moiety.
8. The method of claim 7 , wherein the second forward primer hybridizes to a nucleotide sequence of SEQ ID NO: 3.
9. The method of claim 7 , wherein the second forward primer comprises a nucleotide sequence having at least 80% identity to that of SEQ ID NO: 4; and wherein the second reverse primer comprises the sequence of SEQ ID NO: 6.
10. A kit comprising:
(a) a junction probe comprising
(i) a first region which hybridizes to a sequence within the U3 region of lentiviral nucleic acid 3′ LTR; and
(ii) a second region which hybridizes to a sequence within the R region of lentiviral nucleic acid 3′ LTR; and
(b) a second probe which hybridizes to a TATA-box sequence within 3′ LTR of an HIV nucleic acid;
wherein the first probe and/or the second probe comprises a detectable label.