Real-time RT-PCR for Hepatitis E Virus

Real-time reverse transcriptase-PCR (real-time RT-PCR) as described by Jothikumar et al. [23 (link)] was carried out to detect hepatitis E virus, genotype 3 (HEV3). All real-time RT-PCR reactions were performed in a reaction volume of 16 µL using SensiFAST Probe No-ROX One-Step Kit (Meridian Bioscience, Cincinnati, OH, USA) plus 4 µL (100 ng) template RNA. The reaction was performed at the qTOWER³ G qPCR cycler (Analytik Jena, Jena, Germany). The temperature-time profile applied consists of a reverse transcriptase step of 30 min at 50 °C, followed by an activation step of 15 min at 95 °C and 45 cycles comprising a step of 10 s at 95 °C, followed by a step of 20 s at 55 °C and 15 s at 72 °C [24 (link)].