Table 1.

Commercial RT-PCR kits and thermal cycling conditions used in this study

RT-PCR and manufacturer	PCR reagent sourcea	cDNA step/PCR cycling conditions (45 cycles)b
One-step RT-PCR
Applied Biosystems	AGP kit	45°C for 10 min, 95°C for 10 min/95°C for 15 s, 60°C for 45 s
Invitrogen	SSIII	55°C for 30 min, 94°C for 2 min/94°C for 15 s, 60°C for 60 s
Qiagen	QTRTP kit	50°C for 30 min, 95°C for 15 min/94°C for 15 s, 60°C for 60 s
Two-step RT-PCRc
Applied Biosystems	AGP kit	95°C for 10 mind/95°C for 15 s, 60°C for 45 s
Invitrogen	SSIII	94°C for 2 min/94°C for 15 s, 60°C for 60 s
Qiagen	QTP kit	95°C for 15 min/94°C for 15 s, 60°C for 60 s
Applied Biosystems	TaqMan Universal PCR master mix (ABI)	50°C for 2 min, 95°C for 10 min/95°C for 15 s, 60°C for 60 s

^aAGP kit, AgPath-ID one-step RT-PCR kit; SSIII, SuperScript III one-step RT-PCR system; QTRTP kit, QuantiTect Probe RT-PCR kit; QTP kit, QuantiTect Probe PCR kit.

^bHPeV and EV assays were performed with 5 cycles more than the manufacturers' recommended 40 cycles.

^cTwo-step RT-PCR was performed with cDNA generated using TaqMan reverse transcription reagents (Applied Biosystems).

^dThe initial 94 or 95°C incubation, used with the two-step protocols, activates the hot-start Taq polymerases included in these kits and inactivates the reverse transcriptase in the one-step kits that was not used for cDNA in these protocols.