Detection of Norwalk virus and hepatitis A virus in shellfish tissues with 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 stomach and hepatopancreatic tissues of oysters or hard-shell clams, and viral nucleic acids were purified by a modification of a previously described 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 following advantages compared with the previously described method: (i) more rapid sample processing; (ii) increased test sensitivity; (iii) decreased sample-associated interference with reverse transcription-PCR; and (iv) use of chloroform-butanol in place of the chlorofluorocarbon 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 quantitation of the 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.
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000821:virology; D017250:Caliciviridae Infections / Q000453:epidemiology / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004196:Disease Outbreaks; D005759:Gastroenteritis / Q000453:epidemiology / Q000821:virology; D006507:Hepatovirus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009663:Norwalk virus / Q000235:genetics* / Q000302:isolation & purification*; D010125:Ostreidae / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012313:RNA / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*
D017931:DNA Primers; D004279:DNA, Viral; D012313:RNA