TABLE 2.

Details about the compared real-time PCR assays

AssayCycling structureEnzyme/buffer (source)Primer/probe concn.Detection
Conventional PCR95°C for 5 min, 45 cycles of 95°C for 15 s, 65°C for 15 s, 72°C for 1 min, 72°C for 10 minAmpliTaq Gold/AmpliTaq Gold PCR Master Mix (Applied Biosciences, Foster City, Calif.)0.4 μM of each primer2% agarose gel stained with ethidium bromide
SYBR Green95°C for 15 min, 50 cycles of 95°C for 15 s, 60°C for 1 min, 72°C for 1.5 min, 80°C for 30 s + melting curveHotStar Taq polymerase/QuantiTect SYBR Green PCR Master Mix (QIAGEN, Valencia, Calif.)0.1 μM of each primerFluorescence at the end of the 80°C incubation plateau and during the melting curve
LightCycler95°C for 15 min, 50 cycles of 95°C, 58°C for 10 s,a 72°C for 20 sHotStarTaq polymerase/ QuantiTect Probe PCR Master Mix (QIAGEN, Valencia, Calif.)0.5 μM of each primer/0.1 μM of each probeFluorescence at the end of each annealing plateau
TaqMan1 and TaqMan 295°C for 3 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, 72°C for 30 siTaq DNA polymerase/IQ Supermix (BioRad, Hercules, Calif.)0.5 μM of each primer/0.1 μM of each probeFluorescence at the end of each extension plateau
  • a A touch-down PCR mode was incorporated to stepwise decrease the annealing temperature from 62°C to 58°C during the first eight cycles.