Table 2 PCR oligos and conditions

pol Forward

GGGGATCAAGCCCCACATA

2794 to 3062

2.5 μg DNA

95°C for 20 s followed by 45 cycles of 95°C for 1 s and 60°C for 20 s [14]

   Reverse

GGTGGAGTCTCAGGCAGAAAA

   Probe

[6FAM] TGTTCCAGGGGGACT GGCAAGGTACCAccctgg [DABC]^2,3

pol2 XPOLOF

CCGTGCCCAACCCTTACAACCTCT

2961 to 3330

1.0 μg DNA

40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s for both primary and nested PCR [9]

   XPOLOR

CCGAGGTTCCCTAGGGTTTGTAAT

   XPOLIF

TCCACCCCACCAGTCAGCCTCTCT

   XPOLIR

AAGTGGCGGCCAGCAGTAAGTCAT

   XPOLP

TTGATGAGGCACTGCACAGAGACC

Probe

gag1 GagOF

ATCAGTTAACCTACCCGAGTCGGAC

419 to 1149

0.25 μg DNA; RNA from 62 μL plasma

40 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 45 s for both primary and nested DNA PCR [5, 9]. RT-PCR; Primer 1154R was used for cDNA synthesis at 42°C for 1 hr with the IScript Select cDNA kit (BioRad) followed by 85°C, 5 min to stop the reaction. Nested PCR was then performed as for DNA testing using the Expand High Fidelity PCR System (Roche) and AmpliTaq (Applied Biosystems) for the primary and nested PCRs.

   GagOR

GCCGCCTCTTCTTCATTGTTCTC

   GagIF

GGGGACGAGAGACAGAGACA

   GagOR

CAGAGGAGGAAGGTTGTGCT

   XGagP2

ACCTTGCAGCACTGGGGAGATGTC

Probe

gag2 Forward

AGGTAGGAACCACCTAGTYC

1581 to 1764

RNA from 62 μL plasma

RT-PCR using AgPath-ID one step RT-PCR kit (Applied Biosystems) and BioRad iQ5 iCycler. Reverse primer used for cDNA synthesis at 45°C for 20 min; 95°C for 10 min. 55 cycles at 95°C, 30 s, 52°C, 30 s, 62°C, 30 s.

   Reverse

GTCCTCAGGGTCATAAGGAG

   Probe F

[6FAM]AGCGGGTCTCCAAAACGCGGGC[BHQ1]³

1620

   Probe R

[6FAM]CCTTTTACCTTGGCCAAATTGGTGGGG[BHQ1]³

1673