Reverse transcriptase-polymerase chain reaction detection of hepatitis A virus in naturally contaminated mussels (Mytilus edulis)

With the development of modern molecular techniques, the shellfish enteric viruses became easily detectable. In this study, we evaluated the efficiency of four methods quoted in the literature to extract hepatitis A virus (HAV) from naturally contaminated mussel tissues and to identify the viral material by a sensitive reverse transcriptase-polymerase chain reaction (RT-PCR). The results obtained after real-time RT-PCR showed a decreasing efficiency for HAV mussel extraction with glycine buffer, borate buffer, saline beef-polyvinylpyrrolidone (PVP) and saline beef buffer, respectively, as it was reported in previous works. However, the sensitivity of HAV extraction method using glycine buffer in artificial contaminated mussel was insufficient to allow the detection of HAV at a concentration below 10(2) 50% tissue culture infective doses. Running the real-time amplification reaction with a mixture containing PVP and T4 gene 32 protein has removed RT-PCR inhibitors and has improved sensitivity of this technique in comparison with conventional RT-PCR.