PROTOCOLS
Version APRIL 2024.1
ANRS MIE Virology Network Resistance Study Group
PCR and Sequencing Procedures: HIV-1
Protease
Outer primers :
5’PROT1: 5’-TAA TTT TTT AGG GAA GAT CTG GCC TTC C-3’
3’PROT1: 5’-GCA AAT ACT GGA GTA TTG TAT GGA TTT TCA GG-3’
Inner and sequencing primers :
5’PROT2: 5’- TCA GAG CAG ACC AGA GCC AAC AGC CCC A-3’
3’PROT2: 5’- AAT GCT TTT ATT TTT TCT TCT GTC AAT GGC-3’
Reverse Transcriptase
Outer primers :
MJ3: 5’-AGT AGG ACC TAC ACC TGT CA-3’
MJ4: 5’-CTG TTA GTG CTT TGG TTC CTC T-3’
Inner and sequencing primers:
A35: 5’-TTG GTT GCA CTT TAA ATT TTC CCA TTA GTC CTA TT-3’
NE135: 5’-CCT ACT AAC TTC TGT ATG TCA TTG ACA GTC CAG CT-3’
Other set of primers which can be alternatively used for RT:
Outer primers:
RT18: 5’-GGA AAC CAA AAA TGA TAG GGG GAA TTG GAG G -3’
RT21: 5’- CTG TAT TTC TGC TAT TAA GTC TTT TGA TGG G -3’
Inner and sequencing primers:
RT1: 5’- CCA AAA GTT AAA CAA TGG CCA TTG ACA GA –3’
RT4: 5’- AGT TCA TAA CCC ATC CAA AG –3’
Outer primers :
RT18: 5’-GGA AAC CAA AAA TGA TAG GGG GAA TTG GAG G -3’
JPH4M: 5’-TGA ATG ATT CCT AAT GCA TAY TGT GAG TCT GT -3’
Inner and sequencing primers:
RT1: 5’- CCA AAA GTT AAA CAA TGG CCA TTG ACA GA –3’
PL1M: 5’-CYT GYT TCT GTA TTT CTG CTA YTA AGT CTT TTG -3’
Integrase
Outer primers :
INPS1 : 5’- TAG TAG CCA GCT GTG ATA AAT GTC- 3’
INPR8: 5’-TTC CAT GTT CTA ATC CTC ATC CTG-3’
Inner and sequencing primers :
INPS3 : 5’- GAA GCC ATG CAT GGA CAA G- 3’
INPR9: 5’-ATC CTC ATC CTG TCT ACT TGC C -3’
Other set of primers which can be alternatively used for INT
Outer primers :
HP4149 : 5’-CAT GGG TAC CAG CAC ACA AAG GAA TT- 3’
IN-PCRBD: 5’ –GCT YTC TTT GAA AYA TAC ATA TGR T- 3’
Inner and sequencing primers:
IN-PCRA: 5’-GGA GGA AAT GAA CAA GTA GAT- 3’
IN-SEQ2: 5’-TAC TGC TGT CTT AAG RTG TTC AG- 3’
Other set of primers which can be alternatively used for INT in 2 fragments
Outer primers :
HP4149: 5’-CAT GGG TAC CAG CAC ACA AAG GAA TT- 3’
INPR8: 5’-TTC CAT GTT CTA ATC CTC ATC CTG-3’
Inner and sequencing primers 5’:
IN-PCRA: 5’-GGA GGA AAT GAA CAA GTA GAT- 3’
IN-SEQ2: 5’-TAC TGC TGT CTT AAG RTG TTC AG- 3’
Inner and sequencing primers 3’:
IN-SEQ1: 5’AAA ATT AGC AGG AAG ATG GCC AGT-3’
INPR9: 5’-ATC CTC ATC CTG TCT ACT TGC C -3’
GP41
Outer primers :
1S: 5’-TGGAGGAGGAGATATGAGG-3’
GP2: 5’- ATAATGGTGAATATCCCTGCCTAACTC-3’
Inner and sequencing primers :
ENV3: 5’- TTCCTTGGGTTCTTGGGAGC-3’
2AS: 5’- CTACCAAGCCTCCTACTATC-3’
ENV LONGUE
Outer primers :
ENV31: 5’- CAGTACAATGTACACATGG-3’
GP8-AS: 5’- CCCATAGTGCTTCCTGCT-3’
Inner and sequencing primers :
ENV7: 5’- AATGGCAGTCTAGCAGAAG-3’
CD4BSR4: 5’- TGCTGCTCCCAAGAACCC-3’
Capside :
Outer primers :
6-CA-48F: 5’-MTAGTATGGGCAAGCAGGGA-3’
9-CA-1141R: 5’-TCTTTGCCACARTTGAARCA-3’
Inner and sequencing primers :
7-CA-228F: 5’- RTAARAGACACCAARGAAGC -3’
8-CA-998R: 5’-RCATGCTGTCATCATTTCTT-3’
Other set of primers which can be alternatively used for Capside :
Outer primers :
Capside S1: 5‘-GAS ATA AAA GAC ACC AAG GAA GC-3‘
Capside AS1: 5‘-CAT TTC CAA CAG CCC TTT TTC C-3‘
Inner and sequencing primers :
Capside S2: 5‘- GCA GCA GCT GMC ACA GG-3‘
Capside AS2: 5‘-CCT AAA ATT GCY TCT CTG CAT C-3‘
Conditions for RT, Protease, Integrase and Capsid
RT-PCR Step, using RT-PCR kit Access (PROMEGA):
45’ at 45°C
2’ at 95°C
then: (30’’ at 95°C; 30” at 50°C; 1’ at 68°C) x 40 cycles
7’ at 68°C
Nested PCR step, using Ampli Taq Polymerase with Gene Amp (Roche) :
5’ at 94°C
then (30’’ at 94°C; 30” at 55°C (50°C); 1’ at 72°C) x 45 cycles
7’ at 72°C
Alternative for Nested :
Nested PCR step, using GXL DNA Pol
(10’’ at 98°C; 15” at 55°C; 1’ at 68°C) x 40 cycles
Conditions for RT, Protease, GP41, Env longue, Capsid (set of primer 2) and Integrase
RT-PCR Step, using Transcriptor enzyme (One-Step RT-PCR kit)
| 50°C | 10 min | |
| 94°C | 5 min | |
| 94°C | 10 sec | 35 cycles |
| 50°C | 30 sec | |
| 68°C | 1 min 30 | |
| 68°C | 5 min | |
| 4°C | Hold |
Nested PCR Step, using Promega enzyme (Go Taq G2 hot start polymerase)
| 94°C | 5 min | |
| 94°C | 30 sec | 35 cycles |
| 55°C | 30 sec | |
| 72°C | 1 min | |
| 72°C | 5 min | |
| 4°C | Hold |