A rapid and efficient method for quantitation of genogroups I and II norovirus from oysters and application in other complex environmental samples

The human health risk associated with the consumption of molluscan shellfish grown in sewage-contaminated waters is well established. Noroviruses are the principal agent of shellfish-related illness. This study describes the evaluation of two silica-based viral RNA extraction protocols as well as two real time RT-PCR assays for norovirus detection in shellfish and plankton. Using a GII RNA transcript, the Qiagen RNeasy method was able to recover 80%, 1.85%, and 0.14% of the RNA copies in seeded oyster, small plankton (63–200 μm), and large plankton (>200 μm) samples, respectively, whereas a silica-bead based method was able to recover only 0.175%, 0.0044%, and 0.0006% in the same seeded samples. The detection limit of two published TaqMan RT-PCR assays (A and B) evaluated with RNA run-off transcripts established RT-PCR assay A was more sensitive for detecting low copies of GI.3 RNA whereas RT-PCR assay B was more sensitive for detecting GI.4 and GII.4; however, only assay A was able to detect GI and GII in naturally contaminated shellfish whereas only assay B was able to detect GI and GII in naturally contaminated plankton. The combination of a rapid RNA extraction method followed by both TaqMan RT-PCR assays offers significant advantages for development of routine assays for norovirus detection in bivalve shellfish and shows promise for detection in other high inhibitor environmental sources, such as plankton.