Supplemental Digital Content 1 – Table 1. Primers and thermocycling conditions
For all samples, the complete pol region was initially amplified in two rounds.
Samples successfully amplified were sequenced using two pairs or primers, covering
the protease and RT genes. Samples that did not amplify using Algorithm 1
underwent Algorithm 2, whereby the protease and RT genes were amplified
separately and sequenced separately with two new pairs of sequencing primers.
Nucleic acids were extracted from 200μl of serum using the NucliSens easyMAG
(bioMerieux, The Netherlands) according to manufacturer’s instructions. Primerspecific reverse transcription-PCR was performed on 10 μl of extracted RNA
(OneStep RT-PCR kit from Qiagen, Qiagen, Mississauga, Canada), followed by a
nested PCR reaction (algorithm 1) producing a 1240bp fragment (AmpliTaq Gold,
Applied Biosystems, California, USA). If no product was obtained, two overlapping
PCR products were produced using algorithm 2. All primer sequences and annealing
temperatures are detailed in Table S1.
PCR products were sequenced using BigDye Terminator V3 on an ABI 3130xl
Genetic Analyzer, using 4 primers generating two overlapping regions; sequencing
primers were concordant with the amplified fragment.
Algorithm
Region
amplified
Primer
RT-PCR
GaG1-PR-out.for
RT-new-out.rev
1567bp
TGA ARG AIT GYA CTG ARA GRC AGG CTA AT
CCT CIT TYT TGC ATA YTT YCC TGT T
GaGp6-PR-in.for A
RT-new-in.rev D
1448bp
YTC AGA RCA GRC CRG ARC CAA CAG C
GGY TCT TGR TAA ATT TGR TAT GTC CA
Nested PCR
Algorithm 1
pol
Sequencing
protease
RT-PCR
RT
Algorithm 2
protease
Nested PCR
RT
protease
Sequencing
RT
Size of
product
Sequences (5' → 3')
Sequencing step
GaGp6-PR-in.for
PR-new-in.rev
RT-new-in.for
RT-new-in.rev
53
51.5
YTC AGA RCA GRC CRG ARC CAA CAG C
CTG GTG TYT CAT TRT TKR TAC TAG GT
TTY TGG GAR GTY CAR YTA GGR ATA CC
GGY TCT TGR TAA ATT TGR TAT GTC CA
GaGp1-PR-out.for
PR-new-out.rev
RT-new-out.for
RT-new-out.rev
923bp
GaGp6-PR-in.for
PR-new-in.rev
RT-new-in.for
RT-new-in.rev
835bp
RT-PROT-inner.for
RT-PROT-inner.rev
RT-inner.for
RT-inner.rev
Tm
(°C)
864bp
776bp
TGA ARG AIT GYA CTG ARA GRC AGG CTA AT
AYC TIA TYC CTG GTG TYT CAT TRT T
TTT YAG RGA RCT YAA TAA RAG AAC TCA
CCT CIT TYT TGC ATA YTT YCC TGT T
YTC AGA RCA GRC CRG ARC CAA CAG C
CTG GTG TYT CAT TRT TKR TAC TAG GT
TTY TGG GAR GTY CAR YTA GGR ATA CC
GGY TCT TGR TAA ATT TGR TAT GTC CA
CTT TAR CTT CCC TCA GAT CAC TCT
TCC TGA AGT CTT YAT CTA AGG GAA C
AAT CAG TAA CAG TAC TGG ATG TGG GT
GGC TCT TGA TAA ATT TGA TAT GTC CAT
53
51.4
53
51.4