Comparison and Evaluation of Methods for RNA Extraction of Norovirus from Oyster

Abstract

Norovirus (NV) sometimes causes acute nonbacterial gastroenteritis through contaminated oysters in Japan. RT-PCR reaction or real-time RT-PCR reaction is generally used to detect NV from oysters; however, it is thought that some component in oyster frequently inhibits the PCR reaction. We examined the RNA extraction method for real-time RT-PCR reaction to detect NV: extraction with QIAamp Viral RNA MiniKit after extraction with an equal volume of phenol-chloroform-isoamyl alcohol (PQ method) using 10% homogenized oyster with added NV as a positive control. We tested both genogroups of NV, GI and GIIas positive control, using both domestic and imported oysters in this study. PQ method showed a higher sensitivity (97%) and collection rate compared with the standard method (extraction with QIAamp Viral RNA MiniKit, sensitivity: 57%) or CTAB compound method (extraction with QIAamp Viral RNA MiniKit after processing by the Cetyl trimethyl ammonium bromide method, sensitivity: 88%) irrespective of the genogroup or growing area of the oysters. Our results suggest that the PQ method may effectively remove some PCR inhibiter contained in oysters and is useful in a general laboratory as the extraction method for detection of Norovirus from oyster specimens.