DETECTION OF NORWALK VIRUS AND HEPATITIS-A VIRUS IN SHELLFISH TISSUESWITH THE PCR

A method for the detection of Norwalk virus and hepatitis A virus from shellfish tissues by PCR was developed. Virus was added to the stomac h and hepatopancreatic tissues of oysters or hard-shell clams, and vir al nucleic acids were purified by a modification of a previously descr ibed method (R. L. Atmar, T. G, Metcalf, F. H. Neill, and M. K. Estes, Appl, Environ, Microbiol, 59:631-635, 1993). The new method had the f ollowing advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PC R; and (iv) use of chloroform-butanol in place of the chlorofluorocarb on trichlorotrifluoroethane. In addition, internal standards for both Norwalk virus and hepatitis A virus were made which demonstrated when inhibitors to reverse transcription-PCR were present and allowed quant itation of tile viral nucleic acids present in samples. This assay can be used to investigate shellfish-associated gastroenteritis outbreaks and to study factors involved in virus persistence in shellfish.