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 mu m), and large plankton (> 200 mu m) 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. (c) 2008 Elsevier B.V. All rights reserved.