Critical studies on binding-based RT-PCR detection of infectious Noroviruses

Attempts were made to discriminate between infectious and non-infectious Noroviruses (NoVs) based on their viral binding properties followed by reverse transcription polymerase chain reaction (RT-PCR). Murine norovirus-1 (MNV-1) was employed as a surrogate to test the principle. Detection of both infectious and inactivated MNV-1 was investigated by the plaque assay, RT-PCR and binding-based RT-PCRs. The cell line RAW 264.7 and the ganglioside GD1a were used as binding receptors respectively in combination with RT-PCR. In the second stage of testing, similar approaches were applied to the two main genogroups of human NoVs (GI and GII). Differentiated Caco-2 cells and pig gastric mucin were tested as the binding receptors. Bovine serum albumin (BSA) was used as a non-specific binding control. In this study, the binding-based RT-PCRs decreased the detection of non-infectious NoVs by 1-3-log(10) while all infectious viral particles were detected. No significant difference was observed between the binding-based RT-PCRs within the concentration range investigated, except the binding level of human NoVs GII to pig gastric mucin was higher than to differentiated Caco-2 cells and BSA. This study indicates an improvement in the evaluation of the infectivity of non-cultivable human NoVs. This is also a comprehensive study on both specific and non-specific binding properties of NoVs.