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Duplex RT-qPCR for the detection of hepatitis E virus in water, using a process control

Martin-Latil, Sandra, Hennechart-Collette, Catherine, Guillier, Laurent, Perelle, Sylvie
International journal of food microbiology 2012 v.157 no.2 pp. 167-173
Hepatitis A virus, Hepatitis E virus, Norovirus, RNA, cell culture, detection limit, developed countries, filters, filtration, foods, genome, genotype, hepatitis, humans, mice, pathogens, polymerase chain reaction, process monitoring, rapid methods, reverse transcription, tap water, virion, viruses, water pollution
Human hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water. HEV, in particular genotype III, is currently considered to be an emerging pathogen in industrialized countries. Because of the low infectious dose, an efficient and rapid virus concentration method is required to detect low amounts of HEV in food and water samples for routine control. Because of the absence of a reliable cell culture method for the main enteric viruses most involved in the outbreaks, reverse transcription quantitative real time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food and water samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of the concentration of viral particles, the extraction of nucleic acid and the presence of the potential inhibitors of the RT-qPCR reaction. The aim of this study was to provide a rapid and sensitive method for detecting HEV in water. The method is based on viral concentration by filtration on membrane filters and direct lysis of adsorbed viruses from filters before RNA extraction and RT-qPCR amplification. We developed a one-step duplex RT-qPCR for detecting HEV and the murine norovirus (MNV-1) was used as a process control. The data show that MNV-1 offers a very reliable and simple way of monitoring false-negative results and is a valuable tool in the routine diagnostic laboratory. The limit of detection (LOD) was in the range of 700 to 3500 HEV genome copies/0.5L bottled water and 3500 HEV genome copies/0.5L tap water.