Publication Abstract

Application of RT-PCR to the detection of small round structured viruses in environmentally contaminated molluscan shellfish

Application of RT-PCR to the detection of small round structured viruses in environmentally contaminated molluscan shellfish

J. Green, K. Henshilwood, D.W.E. Brown and D.N. Lees

The Small Round Structured Viruses (SRSV's) are important human pathogens frequently associated with gastroenteritis following consumption of sewage-contaminated molluscan shellfish. Public health controls are hampered by the absence ofmethods for the detection ofthese viruses in shellfish. We have previously described the development of an RT-PCR based assay for the detection of SRS V's in molluscan shellfish. This utilised a sample extraction procedure optimised forremoval of RT-PCR amplification inhibitors. We describe the application ofthese techniques to the detection of SRSV's in shellfish associated with outbreaks of human disease and in random testing of shellfish sold for consumption. Single-round RT-PCR, followed by Southern blot using a pool of four labelled probes, demonstrated that SRSV's could be detected in virtually all oyster samples associated with human disease and with a small percentage of randomly tested samples. However, results were frequently only visualisable through the added sensitivity of Southern blot indicated that the PCR was operating at the limits of sensitivity. This has hindered attempts to sequence positives to confirm result authenticity and genotype. Preliminary data suggest that a nested SRSV PCR approach may overcome these sensitivity limitations and facilitate sample sequencing and other approaches to PCR product characterisation. These developments are discussed in the context of their application to public health controls for molluscan shellfish.

Reference:

J. Green, K. Henshilwood, D.W.E. Brown and D.N. Lees (1997) Application of RT-PCR to the detection of small round structured viruses in environmentally contaminated molluscan shellfish. Proceedings of IAWQ International Symposium on Health related Water Microbiology. Mallorca, 6-10 October 1996. ISBN 9780080433004

J. Green, K. Henshilwood*, D.W.E. Brown and D.N. Lees*

Proceedings of IAWQ International Symposium on Health related Water Microbiology. Mallorca, 6-10 October 1996. ISBN 9780080433004