QuantiTect Multiplex RT-PCR Kit (1000)

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Publication protocol

ERVA-specific singleplex TaqMan® real-time RT-PCR assays targeting G3 VP7, G14 VP7 and NSP3 genes: Primers and probes specific for ERVA G3 VP7 and G14 VP7 were designed as described above (Table 3). The reaction was set up using the QuantiTect™ Probe RT-PCR kit (Qiagen) following the manufacturer’s recommendations. Briefly, the 25 μl reaction contained 12.5 μl of 2X QuantiTect™ Probe RT-PCR Master Mix with ROX, 0.25 μl QuantiTect™ RT Mix, 200 nM TaqMan® fluorogenic probe, 500 nM of each primer, and 5 μl of template RNA (previously subjected to a denaturing step at 95 °C for 5 min). Reverse transcription and amplification were carried out in an ABI 7500 Fast Real-time PCR System (Applied Biosystems®, Life Technologies, Grand Island, NY). The program included 30 min at 50 °C (reverse transcription step), 15 min at 95 °C (PCR initial activation step), followed by 45 cycles at 94 °C for 15 s (denaturation) and 60 °C for 1 min (combined annealing/extension). The NSP3-specific (gene segment 7; pan-rotavirus A) assay was established in the laboratory as previously described (Table 3) [41].

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