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

J Virol Methods. 2000 Oct;90(1):1-14. doi: 10.1016/s0166-0934(00)00174-9.

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.

MeSH terms

  • Animals
  • Bivalvia / virology
  • Hepatovirus / genetics
  • Hepatovirus / isolation & purification
  • Immunoblotting
  • Mamastrovirus / genetics
  • Mamastrovirus / isolation & purification
  • Norwalk virus / genetics
  • Norwalk virus / isolation & purification
  • Ostreidae / virology
  • RNA Viruses / genetics*
  • RNA Viruses / isolation & purification*
  • RNA, Viral / analysis
  • RNA, Viral / isolation & purification
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Sensitivity and Specificity
  • Shellfish / virology*

Substances

  • RNA, Viral