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Submitted to: Meeting Abstract
Publication Type: Proceedings Publication Acceptance Date: 2/22/2004 Publication Date: 6/14/2004 Citation: Richards, G.P. 2004. Molecular methods to detect enteric viruses in molluscan shellfish. Meeting Abstract. Interpretive Summary: Technical Abstract: Enteric virus contamination of molluscan shellfish continues to cause illness throughout the world. The advent of molecular biological methods has enhanced the ability to detect enteric viruses in shellfish. Prior to analysis, viruses must be extracted from shellfish tissues and concentrated. We developed a method to extract and concentrate the RNA from hepatitis A and hepatitis E viruses, poliovirus, and Noroviruses from oysters and clams. The concentrated RNAs are then assayed by reverse-transcription polymerase chain reaction (RT-PCR) and the presence of product signals virus presence. The RT-PCR method works well on the hepatitis viruses which have conserved genomes, but fails to amplify many of the Noroviruses. We developed a real-time RT-PCR procedure which amplifies a wide array of genogroup I and II Noroviruses using broadly reactive primers and SYBR green detection. Product verification is rapid, simple, and is accomplished by means of first derivative melt curve data. Together, the extraction and assay procedures allow the detection and semi-quantitation of Noroviruses from at least 13 different genetic clusters. This real-time RT-PCR technique may also be used on stool preparations in epidemiological investigations to identify the sources of Norovirus transmission. |
