Seminested RT-PCR systems for small round structured viruses and detection of enteric viruses in seafood

Highly sensitive seminested RT-PCR systems for the specific detection of genotype I and II small round structured viruses (SRSVs) were developed based on the nucleic acid information deposited in the databanks. SRSVs could be detected in 107-fold dilutions of three different stool samples. In addition, a rapid and simple purification protocol for enteric viruses from seafood tissues was elaborated using poliovirus (PV) as model. The virus isolation and viral RNA purification include the following steps: elution of the viruses from the seafood tissue with glycine buffer, their concentration by PEG-precipitation, lysis of viral particles with guanidine hydrochloride and viral RNA isolation using a silica based membrane. The detection limit was 3 to 30 TCID50 of poliovirus in 1.25 g of seeded seafood tissues without marked food matrix differences, whereas SRSV viruses were 10- and 100-fold better detected in mussels than in shrimps and oysters, respectively. The newly developed purification method, which was shown to remove potential RT-PCR inhibitors present in mussel tissue samples, was applied in a small market survey. 15 mussels, 15 oysters and 12 shrimps were examined for the presence of Hepatitis A virus (HAV), Enterovirus (EV), Rotavirus (RV) and SRSV using specific RT-PCR detection systems. The finding of three oyster samples positive for Rotavirus demonstrated the successful application of our method for the detection of enteric viruses in naturally contaminated seafood samples. The rapid isolation method might be suitable for application in routine testing laboratories and will help to improve public health controls for seafood.