Viral RNA extraction was carried out on 150 μl of viral suspension using a NucleoSpin® RNA virus kit (Macherey-Nagel GmbH & Co.) according to the manufacturer’s instructions. Primers, probes and RT-qPCR conditions used in this study are listed in Table 1 for HEV and in the ISO 15216:2017 for HAV. Modified-probe included in assay A (Schlosser et al., 2014 (link)) contains a ZEN internal quencher. Modification of assay C (adapted from Mansuy et al., 2004 (link)) consists of an RT reaction held at 45°C for 60 min. RT-qPCRs were carried out in 96-well plates using the LightCycler 480 instrument (Roche Diagnostics) and a half-scale modification of the RNA UltraSense One-Step quantitative RT-PCR system (Invitrogen SA), by using half volumes of all reagents.
Quality control of the RT-qPCR process included negative (nuclease-free water) and positive (RNA) controls added to each PCR plate. Each viral RNA was analyzed in duplicate. HEV and HAV quantification was calculated by plotting the quantification cycles (Cqs) to an external standard curve built with the International Standard WHO HEV RNA (250,000 IU/ml) and HAV reference material (code RM000HAV, Public Health England), respectively.
Quality control of the RT-qPCR process included negative (nuclease-free water) and positive (RNA) controls added to each PCR plate. Each viral RNA was analyzed in duplicate. HEV and HAV quantification was calculated by plotting the quantification cycles (Cqs) to an external standard curve built with the International Standard WHO HEV RNA (250,000 IU/ml) and HAV reference material (code RM000HAV, Public Health England), respectively.
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