Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2

Abstract

Background: Rabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome.

Results: This study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 10² copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2-3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR.

Conclusions: In conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Keywords: GI.2/RHDV2/b; RHDV; Rabbit haemorrhagic disease virus; TaqMan real-time PCR.