A comparison of RT-PCR-based assays for the detection of HAV from shellfish.

The hepatitis A virus (HAV) is the most common cause of viral infection linked to shellfish consumption. The lack of correlation between the fecal coliform indicators and the presence of enteric viruses in shellfish and their harvesting waters points to the need for molecular methods to detect viruses. We compared two RT-PCR based techniques currently available for the detection of the hepatitis A virus (HAV) in shellfish. Both approaches involve extraction of viral particles by glycine buffer and concentration of virus particles by one or two PEG precipitation steps. One procedure involves as RNA extraction method the use of oligo (dT) cellulose to select poly (A) RNA, and the other uses a system in which total RNA is bound on silica membrane. Comparison of the two RT-PCR based methods highlighted the efficiency of the first approach which is less time-consuming and technically demanding than the second.

Detection of hepatitis A virus in shellfish (Mytilus galloprovincialis) with RT-PCR.

A PCR assay for the detection of hepatitis A virus (HAV) in shellfish is described. The procedure involves the concentration of viral particles with the use of polyethylene glycol (PEG), followed by viral RNA extraction and purification with oligo(dT) cellulose. Reverse transcriptase-PCR detection was accomplished in a single step with the use of primers specific for the VP3-VP1 region of the genome. The procedure detected one 50% tissue culture infective dose (0.6 PFU) per 25 g of shellfish homogenate. Heminested PCR was then carried out to verify the specificity of the PCR products. The method was used to detect HAV in shellfish samples from EU categories B and C and to evaluate the quality of shellfish in routine monitoring for HAV in view of the relevant public health implications of this foodborne disease.