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 devel oped based on the nucleic acid information deposited in the databanks. SRSVs could be detected in 10(7)-fold dilutions of three different st ool 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 wi th glycine buffer, their concentration by PEG-precipitation, lysis of viral particles with guanidine hydrochloride and viral RNA isolation u sing a silica based membrane. The detection limit was 3 to 30 TCID50 o f poliovirus in 1.25 g of seeded seafood tissues without marked food m atrix differences, whereas SRSV viruses were 10- and 100-fold better d etected in mussels than in shrimps and oysters, respectively. The newl y developed purification method, which was shown to remove potential R T-PCR inhibitors present in mussel tissue samples, was applied in a sm all market survey. 15 mussels, 15 oysters and 12 shrimps were examined for the presence of Hepatitis A virus (HAV), Enterovirus (EV), Rotavi rus (RV) and SRSV using specific RT-PCR detection systems. The finding of three oyster samples positive for Rotavirus demonstrated the succe ssful application of our method for the detection of enteric viruses i n naturally contaminated seafood samples. The rapid isolation method m ight be suitable for application in routine testing laboratories and w ill help to improve public health controls for seafood. (C) 1997 Elsev ier Science B.V.