Development of a molecular method for the detection of enteric viruses in oysters

Abstract

Detection of enteric virus contamination of shellfish is limited by current methodology, which is time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based on nucleic acid amplification have been hampered by high sample volumes and the presence of enzymatic inhibitors. The goal of this study was to develop methods to purify and concentrate intact virions from oyster extracts to a volume and quality compatible with viral genomic nucleic acid amplification by reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram oyster samples were homogenized and processed by standard adsorption-elution-precipitation methodology and then seeded with 105PFU of poliovirus 1 (PV1) or hepatitis A virus (HAV). Seeded viruses were concentrated by fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform extraction, and cetyltrimethyl ammonium bromide (CTAB) precipitation to a volume of 100 μ1 with removal of RT-PCR inhibitors. Virus recovery after elution of PEG precipitates was 50% for PV1 and 15 to 20% for HAV as evaluted by cell culture infectivity. The CTAB precipitation step yielded aconcentrated sample which was directly compatible with RTPCR reactions and capable of detecting about 100 placque=forming units (PFU) of PV1 or HAV. When 50-g oyster extracts were seeded and processed by the entire concentration and purification scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 104 PFU of HAV and 103 PFU of PV1, with recoveries of 1 to 5% of seeded viruses. Copyright©, International Association of Milk, Food and Environmental Sanltarians.