Improved method for rotavirus detection in oysters using RT-PCR: Suitability of a commercial PCR kit

Commercially marketed kits are now available for PCR reactions. This study was conducted to determine the suitability of one of these kits (PCR supermix kit from Life Technologies Inc) for use in environmental testing for rotavirus in commercially cultured oysters. We focused on developing a rapid, efficient and inhibitor-free oyster-processing procedure which could be used for sensitive viral genome amplification by reverse transcripts PCR (RT-PCR) in raw oysters using a commercial kit for genome amplification, Rotavirus SA11 strain was used to evaluate the efficiency of virus recovery. Oyster tissues were seeded with 3 x 10(7) ffu of rotavirus. Oyster-processing included elution in TPB (Tryptone phosphate broth/glycine (pH 9.0), virus precipitation using polyethylene glycol. sonication, and RNA extraction. RNA extraction methods evaluated included CTAB/SDS followed by phenol/ chloroform extraction (standard method), or the commercial reagent TRIZOL LS(TM) Both were equally effective for removal of PCR inhibitors. Detection limit of the method described in this study was 0.03 ffu rotavirus SA11 recovered from the oyster tissues. We conclude that the Supermix((R)) commercial PCR kit can provide a more rapid and sensitive alternative for virus detection in oysters when compared to traditional PCR protocols. This is especially beneficial when large numbers of environmental samples require analysis.