Collaborative evaluation of a method for the detection of Norwalk virus in shellfish tissues by PCR

A multicenter, collaborative trial was performed to evaluate the reliability and reproducibility of a previously described method for the detection of Norwalk virus in shellfish tissues with the PCR (R.L. Atmar, F. H. Neill, J. L. Romalde, F. Le Guyader, C. M. Woodley, T. G. Metcalf, and M. K. Estes, Appl. Environ. Microbiol. 61:3014-3018, 1995). Virus was added to the stomachs and hepatopancreatic tissues of oysters or hard-shell clams in the control laboratory, the samples were shipped to the participating laboratories, and viral nucleic acids were extracted and then detected by reverse transcription-PCR. The sensitivity and specificity of the assay were 85 and 91%, respectively, when results were determined by visual inspection of ethidium bromide-stained agarose gels; the test sensitivity and specificity improved to 87 and 100%, respectively, after confirmation by hybridization with a digoxigenin-labeled, virus-specific probe. We have demonstrated that this method can be implemented successfully by several laboratories to detect Norwalk virus in shellfish tissues.