Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR

Appl Environ Microbiol. 1995 Aug;61(8):3014-8. doi: 10.1128/aem.61.8.3014-3018.1995.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Bivalvia / virology
  • Caliciviridae Infections / epidemiology
  • Caliciviridae Infections / virology
  • DNA Primers / genetics
  • DNA, Viral / genetics
  • Disease Outbreaks
  • Gastroenteritis / epidemiology
  • Gastroenteritis / virology
  • Hepatovirus / genetics*
  • Hepatovirus / isolation & purification*
  • Humans
  • Molecular Sequence Data
  • Norwalk virus / genetics*
  • Norwalk virus / isolation & purification*
  • Ostreidae / virology
  • Polymerase Chain Reaction / methods*
  • Polymerase Chain Reaction / standards
  • Polymerase Chain Reaction / statistics & numerical data
  • RNA / standards
  • Reference Standards
  • Sensitivity and Specificity
  • Shellfish / virology*

Substances

  • DNA Primers
  • DNA, Viral
  • RNA