Detection of enterovirus and hepatitis A virus RNA in mussels (Mytilus spp.) by reverse transcriptase-polymerase chain reaction

Aims: a simple and effective method of concentrating and purifying enteric viruses from mussel samples to be detected by nucleic acid extraction reverse transcriptase-polymerase chain reaction (RT-PCR) has been evaluated. Methods and Results: Seeded mussels were processed by alkaline elution, polyethylene glycol (PEG) precipitation, solvent extraction and PEG precipitation. Final concentrates were assayed by infectivity and RT-PCR after nucleic acid extraction. Two RNA extraction methods were comparatively evaluated for removing inhibitory substances: guanidinium thiocyanate extraction and Purescript(TM) RNA isolation. Both procedures removed most inhibitors allowing the detection of viral RNA at inoculum levels as low as 4 pfu g(-1) for poliovirus type 1 and 1.8-18 most probable number of cytopathogenic units g(-1) for HAV, When inhibitors remained, they were efficiently removed by Sephadex column chromatography before RNA extraction. Conclusions: The described method is effective for the detection of enteric viruses in mussels by RT-PCR. The use of Purescript(TM) RNA isolation makes the method faster, safer and very easy to perform.