Table 2

Summary of laboratory DNA amplification, hybridization, and detection methodsa

AssayPrimersPCR cycles, temperatures, and timeProbesReference
CommonSV5/SV6Denaturing at 95°C for 5 minSV.probe (16)
45 amplifications at 95°C for 1 min, 59°C for 1 min, 72°C for 1 min
Extension at 72°C for 10 min
2.1PYV.for/SV.revDenaturing at 94°C for 3 minEthidiumSee text
 then (seminested)30 amplifications at 94°C for 1 min, 52°C for 1 min, bromide gel
 SV.for3/SV.rev72°C for 1 min
Extension at 72°C for 5 min
Then
Denaturing at 94°C for 3 min
30 amplifications at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min
Extension at 72°C for 5 min
3.1SV.for2/SV.revDenaturing at 94°C for 4 minSV.probe (17)
50 amplifications at 94°C for 45 s, 55°C for 45 s, 72°C for 45 s
Extension (none)
3.2LA1/LA2Denaturing at 94°C for 4 minVP1.probe (17)
50 amplifications at 94°C for 45 s, 52°C for 45 s, 72°C for 45 s
Extension (none)
5.1RA1/RA2Initial at 94°C for 2 minEthidium (9)
29 amplifications at 94°C for 15 s, 63°C for 15 s, 72°C for 15 s bromide gel
Extension at 72°C for 7 min
Add 1 aliquot of enzyme
Repeat (total = 62 cycles)
5.2SV.for3/SV.revInitial at 94°C for 2 minEthidium (7)
29 amplifications at 94°C for 15 s, 63°C for 15 s, 72°C for 15 s bromide gel
Extension at 72°C for 7 min
Add 1 aliquot of enzyme
Repeat (total = 62 cycles)
7.1SV.for3/SV.revDenaturing at 94°C for 5 minSV1.probe (13 , 39)
45 amplifications at 95°C for 1 min, 59°C for 1 min, 72°C for 1 min
Extension at 72°C for 10 min
8.14FG-6BGDenaturing at 94°C for 5 minP5 (42)
39 amplifications at 94°C for 1 min, 50°C for 2 min, 72°C for 1 min
Extension at 72°C for 6 min
9.1Southern blotSouthern blot without PCR amplificationSV40FL (6)
  • a One laboratory used Southern blot without PCR amplification; all others used PCR. The eight PCR laboratories agreed to test specimens using a Common PCR assay. Five of the eight PCR laboratories also used assays that they have reported previously in the literature (as referenced). The PCR assay in Lab 2 had not been published, and it is described in the text.