Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR

Abstract

Comparison of two viral extraction methods in order to establish a sensitive and simple detection method for human noroviruses (NV) in bivalve shellfish. A direct RNA extraction method and an alkaline virus elution-concentration method were tested on artificially contaminated mussels. The latter used an alkaline buffer and polyethylene glycol (PEG) to isolate and concentrate the virus particles from shellfish. In both methods Trizol was used to release RNA. The final RNA extracts were amplified and detected with conventional and real-time reverse transcriptase PCR. The direct RNA extraction method was not able to detect low inoculation levels. However, the virus elution-concentration method was more sensitive. The alkaline elution-PEG concentration method followed by Trizol effectively removed inhibitory components and fulfilled the demands to be a useful tool for routine testing of shellfish for NV detection. Because of the lack of standardized methods to detect NV in shellfish, many 'in-house' extraction methods are used in practice. A comparison of these methods aims to determine a simple, rapid and sensitive method that could be a candidate method for screening suspected shellfish.