Development of a PCR-based method for the detection of enteroviruses and hepatitis a virus in molluscan shellfish and its application to polluted field samples¹

The use of the polymerase chain reaction (PCR) for detection of low levels of enteric viruses in bivalve shellfish is hindered by the presence of potent amplification inhibitors. A procedure previously developed for removing the majority of these amplification inhibitors is applied to the detection of enteroviruses and hepatitis A virus in naturally polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly polluted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcription (RT)-PCR results were compared with conventional enterovirus isolation for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. At one field site shellfish were positive by enterovirus RT-PCR but negative for virus isolation. All shellfish tested were negative for hepatitis A by RT-PCR. The procedure for removal of PCR amplification inhibitors should be equally applicable to the detection of Norwalk and related Small Round Structured Viruses (SRSVs) in shellfish.