Development of a Molecular Method for the Detection of Enteric Viruses in Oysters

摘要: Detection of enteric virus contamination shellfish is limited by current methodology, which time-consuming, tedious, and lacking in sensitivity due to reliance on cell culture infectivity. Alternative detection methods based nucleic acid amplification have been hampered high sample volumes the presence enzymatic inhibitors. The goal this study was develop purify concentrate intact virions from oyster extracts a volume quality compatible with viral genomic reverse transcriptase-polymerase chain reaction (RT-PCR). Fifty-gram samples were homogenized processed standard adsorption-elution-precipitation methodology then seeded 10 5 PFU poliovirus 1 (PV ) or hepatitis A (HAV). Seeded viruses concentrated fluorocarbon extraction, polyethylene glycol (PEG) precipitation, chloroform cetyltrimethyl ammonium bromide (CTAB) precipitation 100 μl removal RT-PCR Virus recovery after elution PEG precipitates 50% for PV1 15 20% HAV as evaluted CTAB step yielded directly reactions capable detecting about placque=forming units (PFU) PVI HAV. When 50-g entire concentration purification scheme, direct RNA possible at initial inoculum levels 4 3 PVI, recoveries 5% viruses.