Abstract
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. 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 Purescripttrade mark 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. The described method is effective for the detection of enteric viruses in mussels by RT-PCR. The use of Purescripttrade mark RNA isolation makes the method faster, safer and very easy to perform.
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