Development of monoclonal antibody-coated immunomagnetic beads for separation and detection of norovirus (genogroup II) in faecal extract samples

摘要

The aim of this study is to develop an RT-PCR assay combined with immunomagnetic beads (IMS/RT-PCR) coating monoclonal antibody (Mab) for separation and detection of norovirus (genogroup II) in faecal samples. We furthermore compare its detection limits with IMS/RT-PCR using polyclonal antibody (Pab) and the TRIzol extraction method followed by RT-PCR (TRIzol-RT-PCR). Mab-coated beads and Pab-coated beads were added to a series of tenfold dilutions of faecal extract containing norovirus in 1 ml PBS. After incubation and collection, the RNA was released by heating from virus separated by beads. The tenfold dilutions of faecal were also extracted with TRIzol reagent. The RNA was used as the template for RT-PCR detection (primers: JV12-JV13). IMS/RT-PCR using Mab showed an endpoint in the 10" dilution and was 10po times more sensitive than IMS/RT-PCR using Pab and was at least 10pd times more sensitive than TRIzol-RT-PCR method. IMS/RT-PCR using Mab proved to be a more sensitive method of noroviruses (NVs) detection than IMS/RT-PCR using Pab and the TRIzol-RT-PCR method. This is the first study to detect NVs with IMS/RT-PCR using Mab, and could serve as a model for future assays when broadly reactive NVs-specific Mabs are developed.