Zhou Jun, Ma Yanjun, Wang Min, Zhang Yi, Chen Bin, Chen Dishi, Li Li, Li Mingxiang
Sichuan BoCe Testing Tech Co., Ltd., Chengdu 610041, China.
Center for Animal Disease Control and Prevention, Bazhou District, Bazhong, Sichuan Province 636600, China.
J Virol Methods. 2022 Jun;304:114526. doi: 10.1016/j.jviromet.2022.114526. Epub 2022 Mar 24.
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.
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 10 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.
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.
兔出血症（RHD）是由杯状病毒（兔杯状病毒属）引起的欧洲兔（穴兔）的一种高度传染性急性致命性肝炎。截至2010年，分离出的所有兔出血症病毒（RHDV）都属于一个基因型。2010年，一种新的RHDV基因型（RHDV2/b，根据系统发育分析目前命名为GI.2）在法国出现。本研究的目的是开发一种快速、简单、特异且灵敏的TaqMan实时荧光定量PCR检测方法，用于检测经典株RHDV和RHDV2。针对病毒基因组保守区域内的RHDV和RHDV2的VP60基因设计了特异性引物和探针。
本研究证明对RHDV和RHDV2具有高度特异性，与其他非目标病毒无交叉反应。本研究的检测限分别为RHDV和RHDV2的10个拷贝。该检测方法的批内和批间变异系数均小于5%。使用质粒评估该方法的重现性，获得的变异系数为0.2 - 3.70。在79份临床样本中，68份为阳性样本（86.08%），其中60份为经典RHDV变异株（75.9%），4份为共感染（5.06%），8份为RHDV2（10.12%），与传统RT-PCR相比，这些结果更具敏感性。
总之，基于RHDV和RHDV2的VP60基因的这种双重TaqMan RT-qPCR可成为RHDV和RHDV2诊断及分子流行病学研究的有价值工具。
