牛诺如病毒实时荧光定量PCR检测方法的建立及应用

牛诺如病毒（BNoV）是国内新发的犊牛腹泻病原，笔者拟建立检测BNoV的Real-time PCR方法。根据BNoV流行株的RNA聚合酶（RdRp）基因序列设计引物，通过优化反应条件和体系，成功建立基于EvaGreen检测BNoV的Real-time PCR方法。该检测方法的Ct值与标准品模板在2.24×10²~2.24×10⁸拷贝·μL^-1线性关系良好，相关系数R²=0.997，扩增效率为98.44%；该方法可特异性检出BNoV，对其他犊牛腹泻相关病原呈阴性；最低检测限为22.4拷贝·μL^-1；批间和批内的变异系数均小于2%，重复性好。对2017年9月-2019年5月采自河南省的221份犊牛腹泻样本中BNoV的检出率为11.31%（25/221），采样场阳性率为92.86%（13/14）。本试验所建方法灵敏度高、特异性强、稳定性好，为BNoV的检测和流行病学调查提供了有力手段。

Establishment and Application of a Real-time PCR Assay for Detecting Bovine Norovirus

Bovine norovirus (BNoV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time PCR assay for detecting BNoV. One pair of primers was designed based on RNA-dependent RNA polymerase (RdRp) gene of BNoV. The EvaGreen real-time PCR assay was successfully developed after the optimization of amplification conditions. The test results showed that the Ct value showed a good linear relationship with the standard in the range of 2.24×10²-2.24×10⁸copies·μL^-1 and the correlation coefficient R²=0.997, and the amplification efficiency was 98.44%. There is no specific amplification of other common calf diarrhea pathogens, only BNoV were positive. The detection limit of the method was 22.4 copies·μL^-1 for BNoV. The inter-assay and the intra-assay coefficient of variation were both less than 2%, indicating a good repeatability. 221 clinical samples that collected from the diarrheic calves in Henan Province during September 2017 to May 2019 were detected using this real-time PCR assay, and the BNoV detection rate was 11.31% (25/221), the farms positive rate was 92.86% (13/14). These results indicated that the real-time PCR assay has good sensitivity, specificity and repeatability, which can be provide an effective means for detection and epidemiological investigation of BNoV.