REVERSE-TRANSCRIPTASE PCR DETECTION OF ASTROVIRUS, HEPATITIS-A VIRUS,AND POLIOVIRUS IN EXPERIMENTALLY CONTAMINATED MUSSELS - COMPARISON OFSEVERAL EXTRACTION AND CONCENTRATION METHODS

Four methods of extraction and three methods of concentration of three enteric viruses from mussels were comparatively evaluated by reverse transcriptase PCR (PZT-PCR). Shellfish were experimentally contaminate d by immersion in seawater seeded with astrovirus, hepatitis A virus, or poliovirus. Sixty-gram samples of mussel tissues mere processed by using berate buffer, glycine solution, saline beef, and saline beef-Fr eon extraction methods, The viruses were concentrated by precipitation with polyethylene glycol 6000 (PEG 6000) or PEG 8000 or by organic fl occulation, RT-PCR was performed with RNA extracts from crude shellfis h extracts and concentrates with and without Sephadex LH20 filtration. The glycine solution and berate buffer extraction methods resulted in significantly more RT-PCR-positive samples than the saline beef extra ction method. We assessed the efficiency of 20 combinations of extract ion and concentration methods. The berate buffer-organic flocculation, berate buffer-PEG 6000, and glycine solution-PEG 6000 combinations ga ve RT-PCR-positive results for all 27 samples analyzed for the three v iruses. Detoxification of the samples by Sephadex LH20 filtration sign ificantly decreased the efficiency of RT-PCR virus detection.