Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR

Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure based on the enzymatic liquefaction of shellfish digestive tissues without any mechanical homogenisation step, followed by a simple clarification of the lysate using dichloromethane extraction, was developed. Viral RNA is isolated directly from the shellfish extract by a guanidium thiocyanate-silica extraction method, adapted for the use of a vacuum manifold system. Virus-specific RT-PCR assays were set up for detection of genomic sequences of the predominant viral pathogens, HAV, Astrovirus and Norwalk-like viruses (from genogoups I or II). The specificity of the amplicons is confirmed finally by hybridisation with DIG-labelled specific probes. The overall procedure applied to shellfish samples spiked with HAV particles allowed a detection of 20 pfu of HAV per g of hepatopancreas. In addition, up to 20 samples can be tested within 24 h. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Virological Methods Elsevier

Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR

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Publisher
Elsevier
Copyright
Copyright © 2000 Elsevier Science B.V.
ISSN
0166-0934
eISSN
1879-0984
DOI
10.1016/S0166-0934(00)00174-9
Publisher site
See Article on Publisher Site

Abstract

Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure based on the enzymatic liquefaction of shellfish digestive tissues without any mechanical homogenisation step, followed by a simple clarification of the lysate using dichloromethane extraction, was developed. Viral RNA is isolated directly from the shellfish extract by a guanidium thiocyanate-silica extraction method, adapted for the use of a vacuum manifold system. Virus-specific RT-PCR assays were set up for detection of genomic sequences of the predominant viral pathogens, HAV, Astrovirus and Norwalk-like viruses (from genogoups I or II). The specificity of the amplicons is confirmed finally by hybridisation with DIG-labelled specific probes. The overall procedure applied to shellfish samples spiked with HAV particles allowed a detection of 20 pfu of HAV per g of hepatopancreas. In addition, up to 20 samples can be tested within 24 h.

Journal

Journal of Virological MethodsElsevier

Published: Oct 1, 2000

References

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