The development of polymerase chain reaction assays for detection of smallround structured and other human enteric viruses in molluscan shellfish

Public health controls for molluscan shellfish are hampered by the absence of methods for the detection in shellfish of the viral pathogens causing il lness. The polymerase chain reaction (PCR) offers a potential way forward; however, its application to such complex samples as shellfish is hindered b y the presence of potent amplification inhibitors. We describe the developm ent of a procedure employing a combination of virus purification and nuclei c acid extraction for the removal of the majority of such PCR inhibitors fr om shellfish. Initial developmental work used seeded poliovirus as a model and showed that PCR sample tolerance ranged from 2 to 5 g shellfish for hig hly polluted samples with over-all sensitivity limits of <10 plaque-forming units (PFU) poliovirus. These methods have recently been applied to the de tection of hepatitis A virus and Norwalk and related small round structured viruses (SRSVs) in shellfish. Initial seeding experiments have shown that the method for removal of amplification inhibitors is equally applicable to the detection of these viruses by PCR in shellfish. However a single round reverse transcriptase-polymerase chain reaction (RT-PCR) proved insufficie ntly sensitive to reliably detect SRSVs in shellfish samples associated wit h incidents of human infection. We describe the further development of a ne sted RT-PCR procedure for the detection of SRSVs in shellfish and the appli cation of this assay for the detection of SRSV in commercially produced she llfish and in shellfish implicated in outbreaks of gastroenteritis. These s tudies show that detection of human enteric viruses in molluscan shellfish by PCR is feasible and may ultimately contribute to the further development of public health controls.