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ABSTRACT: Currently, hepatitis A virus (HAV) detection is not yet included in the regulation provided for food safety controls. However,
the 2073/2005/EC regulation on microbiological criteria applied to food takes into account the sanitary issue as to viral
contamination of food but postpones the definition of specific criteria for pathogenetic virus detection until reliable analytical
methods are available. In this study, a PCR-based method for detection of HAV was subjected to a validation process in order
to describe its performance and to check its reliability for shellfish monitoring. Shellfish were processed for virus recovery
through PEG8000-mediated sedimentation. Viral RNA extraction was carried out with a commercial kit and extracted RNA was then
subjected to a reverse transcriptase heminested PCR (rt-heminested PCR). Foetal rhesus monkey kidney (FRhK4) cell culture
infected with HAV worked as positive control in the assay: so, also supernatant from infected FRhK4 cells was subjected to
a parallel validation process, starting to RNA extraction. The validation process was carried out by establishing qualitative
and semi-quantitative parameters concerning accuracy, precision, analytical specificity and sensitivity (limit of detection,
LOD), robustness and stability. The HAV detection method for shellfish and supernatant from infected FRhK4 cells showed 100%
accuracy and precision. It has proved robust and stable; the LOD of rt-heminested PCR was estimated as nine genomic copies
of DNA target. The validation process has objectively demonstrated that the HAV detection method on shellfish has good performance,
so it might be applied with enough reliability in food safety official controls.
Food and Environmental Virology 04/2012; 1(2):97-104. · 1.56 Impact Factor
