Primers and probes for the amplification and detection of HIV Gag, Rev and Nef polynucleotides
The invention relates to improved methods and compositions for the nucleic acid amplification of one or multiple variants (strains) of Human Immunodeficiency Virus (HIV) present in a sample, and preferably in a sample from a pathogen infected individual. In particular, novel primers, methods and kits for the amplification of one or more species of HIV Rev, Gag and Nef nucleic acids are provided. The amplified HIV nucleic acid can be used to identify and/or quantitate HIV variants present in a sample. Nucleic acids produced by the methods of the invention or the proteins encoded thereby can also be used directly as vaccines or to transfect/load antigen presenting cells. The loaded antigen presenting cells can be used as a vaccine for the treatment or prevention of HIV infection.
1. A composition comprising at least one or a combination of Rev T7 promoter primers selected from the group consisting of:
(SEQ ID NO: 24)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCACC
TCCCAACC,
(SEQ ID NO: 25)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCACC
TCCCAGCC,
(SEQ ID NO: 26)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCACC
TCCCAGTT,
(SEQ ID NO: 27)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCTTA
CCCCAACC,
(SEQ ID NO: 28)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCTTA
CCCCAAAC,
(SEQ ID NO: 29)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGGACCCTTA
CCCCAAGC,
(SEQ ID NO: 17)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
ACCT,
(SEQ ID NO: 18)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
GCCT,
(SEQ ID NO: 19)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
TCCT,
(SEQ ID NO: 20)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
ATAC,
(SEQ ID NO: 21)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGATCC
ATAC,
(SEQ ID NO: 22)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
TTAC,
and
(SEQ ID NO: 23)
N 1 N 2 N 3 N 4 N 5 TAATACGACTCACTATAGGN 6 AGACCACCATGTCAGACCC
CTAC;
wherein N 6 is G, A, C or T; and either N 1 is G, N 2 is A, N 3 is A and N 4 and N 5 are T or each of N 1 through N 5 are absent.
2. The composition of claim 1 , wherein N 1 is G, N 2 is A, N 3 is A and N 4 and N 5 are T.
3. The composition of claim 1 , wherein each of N 1 through N 5 are absent.
4. The composition of claim 1 , wherein N 6 is A, C or T.
5. The composition of claim 1 , wherein N 6 is G.
6. The composition of claim 1 , which comprises at least two primers selected from the group consisting of SEQ ID NO:24; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
7. The composition of claim 6 , which comprises SEQ ID NO:24; SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29.
8. The composition of claim 6 , further comprising a Rev 64T reverse primer selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or any combination thereof.
9. The composition of claim 1 , which comprises at least two primers selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
10. The composition of claim 9 , which comprises SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23.
11. The composition of claim 9 , further comprising a Rev 64T reverse primer selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or any combination thereof.
12. A method for amplifying an HIV Rev nucleic acid, comprising carrying out a polymerase chain reaction using at least one or a combination of Rev T7 7912 forward primers selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ CD NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29; and at least one or a combination of Rev R8300 64T reverse primers selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO: 16.