A VIRION CONCENTRATION METHOD FOR DETECTION OF HUMAN ENTERIC VIRUSES IN OYSTERS BY PCR AND OLIGOPROBE HYBRIDIZATION

This article reports the development of a method to purify and concent rate intact virions from oyster extracts to a volume and quality compa tible with viral genomic nucleic acid amplification by reverse transcr iptase PCR (RT-PCR) and confirmation by oligonucleotide probe hybridiz ation, Fifty-gram oyster samples were processed by an adsorption-eluti on-precipitation method and then seeded with 10(1) to 10(5) PFU of pol iovirus type 1 (PV1) or hepatitis A virus (HAV). Seeded viruses in oys ter extracts were purified by fluorocarbon extraction and concentrated by polyethylene glycol (PEG) precipitation and elution, Virus recover y after elution of PEG precipitates was dependent upon PEG concentrati on and averaged 60% for PV1 and 40% for HAV, The next processing step used the protein-precipitating agent Pro-Cipitate (Affinity Technology , Inc., Brunswick, N.J.) in an adsorption-elution-precipitation scheme to further concentrate viruses and reduce sample volumes to 100 mu l. Oyster extracts processed by Pro-Cipitate adsorption-elution-precipit ation were directly compatible with RT-PCR and yielded virus recoverie s of > 80% for both PV1 and HAV, When extracts from 50-g oyster sample s were seeded and processed by the combined concentration and purifica tion scheme, direct RT-PCR detection of viral genomic RNA was possible at initial inoculum levels of 10 PFU for both PV1 and HAV and with lo w levels of Norwalk virus. Virus recoveries based on cell culture infe ctivity were 25 to 35% for PV1 and 5 to 10% for HAV. When tested on ar tificially contaminated raw oysters, the combined method successfully detected greater than or equal to 10(3) PFU of PV1 and HAV and 10(5) R T-PCR-amplifiable units of Norwalk virus, Virus detection by RT-PCR an d cell culture infectivity was consistent and well correlated among re plicate samples and at different virus titers, The procedure developed in this study is rapid, sensitive, and effective for the direct detec tion of enteric viruses in oysters by RT-PCR.