Development of a Virus Elution and Concentration Procedure for Detecting Norovirus in Oysters

Abstract

Low levels of virus contamination and naturally occurring reverse transcription-polymerase chain reaction (RTPCR) inhibitors restrain virus detection in oysters. A rapid and efficient oyster-processing procedure that can be used for sensitive virus detection in oysters was developed. Poliovirus type I Sabin strain was used to evaluate the efficacy of virus recovery. The procedure included (a) acid-adsorption and elution with buffers (0.25 M glycine-0.14 M NaCl, pH 7.5; 0.25 M threonine-0.14 M NaCl, pH 7.5); (b) polyethylene glycol (PEG) precipitation; (c) resuspension in Tween 80/Tris solution and chloroform extraction; (d) the second PEG precipitation; (e) viral RNA extraction with TRIzol and isopropanol precipitation; and (f) RT-PCR combined with semi-nested PCR. The overall recovery of elution/concentration was 19.5% with poliovirus. The whole procedure usually takes 19 hr. The overall detection sensitivity was 4 RT-PCR units of genogroup I norovirus (NoV) and 6.4 RT-PCR units of genogroup II Nov/25 g of oysters initially seeded. The virus-detecting method developed in this study should facilitate the detection of low levels of NoV in oysters.