DEVELOPMENT OF A PCR-BASED METHOD FOR THE DETECTION OF ENTEROVIRUSES AND HEPATITIS-A VIRUS IN MOLLUSCAN SHELLFISH AND ITS APPLICATION TO POLLUTED FIELD SAMPLES

The use of the polymerase chain reaction (PCR) for detection of low le vels of enteric viruses in bivalve shellfish is hindered by the presen ce of potent amplification inhibitors. A procedure previously develope d for removing the majority of these amplification inhibitors is appli ed to the detection of enteroviruses and hepatitis A virus in naturall y polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly pollu ted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcript ion (RT)-PCR results were compared with conventional enterovirus isola tion for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. A t one field site shellfish were positive by enterovirus RT-PCR but neg ative for virus isolation. All shellfish tested were negative for hepa titis A by RT-PCR. The procedure for removal of PCR amplification inhi bitors should be equally applicable to the detection of Norwalk and re lated Small Round Structured Viruses (SRSVs) in shellfish.