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Comparison of two extraction methods for the detection of hepatitis A virus in semi-dried tomatoes and murine norovirus as a process control by duplex RT-qPCR

Martin-Latil, Sandra, Hennechart-Collette, Catherine, Guillier, Laurent, Perelle, Sylvie
Food microbiology 2012 v.31 no.2 pp. 246-253
Hepatitis A virus, Norovirus, RNA, cell culture, detection limit, epidemiological studies, foodborne illness, hepatitis, mice, polymerase chain reaction, process monitoring, reverse transcription, salads, small fruits, tomatoes, ultracentrifugation, virion, viruses, Australia, France, Netherlands
Enteric viruses are important agents of foodborne diseases. Due to their low infectious doses and low concentrations in food samples, an efficient and rapid virus concentration method is required for routine control. Because of the absence of a reliable cell culture method for most of the enteric viruses involved in outbreaks, reverse transcription quantitative real-time PCR (RT-qPCR) is now widely used for the detection of RNA viruses in food samples. One of the general requirements for viral diagnosis concerns the use of a process control to monitor the efficiency of viral particle concentration, nucleic acid extraction and the presence of potential inhibitors of the RT-PCR reaction. Recent epidemiological studies have linked hepatitis A outbreaks to the consumption of semi-dried tomatoes (SDT) in Australia, the Netherlands and France. In this study, the virus concentration reference method proposed by the CEN/TC275/WG6/TAG4 working group for samples of soft fruit and salad vegetables was compared to a method including an ultracentrifugation step to recover hepatitis A virus (HAV) in SDT. Murine norovirus (MNV-1) was used as a process control and detected simultaneously with HAV in a one-step duplex RT-qPCR in both procedures. The LOD of HAV was 10 PFU and 1 PFU of HAV/25 g of SDT in the presence or absence of MNV-1 respectively, whatever the method used. We conclude that both methods achieved an identical limit of detection and that the MNV-1 offers a very reliable and simple way to monitor the quality of the extraction procedures and the presence of RT-qPCR inhibitors.