牛诺如病毒实时荧光定量PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2020.07.025

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (7): 1728-1736.doi: 10.11843/j.issn.0366-6964.2020.07.025

牛诺如病毒实时荧光定量PCR检测方法的建立及应用

师志海^1,3, 王文佳², 兰亚莉^1*, 张彬¹, 孟红丽¹, 王亚州¹, 滑留帅¹, 徐照学^1,3*

1. 河南省农业科学院畜牧兽医研究所, 郑州 450002;
2. 河南牧业经济学院动物医药学院, 郑州 450046;
3. 河南省畜禽繁育与营养调控重点实验室, 郑州 450002

收稿日期:2020-01-02 出版日期:2020-07-25 发布日期:2020-07-22
通讯作者: 兰亚莉,主要从事兽医临床工作,E-mail:yali2005haonan@sina.com;徐照学,主要从事牛产业发展工作,E-mail:xuzhaoxue11@163.com
作者简介:师志海(1982-),男,河南辉县人,副研究员,博士,主要从事兽医临床与病毒学研究,E-mail:szhvet@163.com;王文佳(1987-),女,河南平顶山人,助教,硕士,主要从事动物病原分子生物学研究,E-mail:wangwenjia052@163.com。
基金资助:
河南省重点研发与推广专项（192102110074）；国家肉牛牦牛产业技术体系（CARS-37）；国家重点研发计划（2018YFD051700）

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

SHI Zhihai^1,3, WANG Wenjia², LAN Yali^1*, ZHANG Bin¹, MENG Hongli¹, WANG Yazhou¹, HUA Liushuai¹, XU Zhaoxue^1,3*

1. Institute of Animal Husbandry and Veterinary Medicine, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
2. College of Veterinary Medicine and Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
3. Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation, Zhengzhou 450002, China

Received:2020-01-02 Online:2020-07-25 Published:2020-07-22

摘要/Abstract

摘要： 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。

关键词: 牛诺如病毒, 实时荧光定量PCR, 犊牛腹泻, 检测

Abstract: 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.

Key words: bovine norovirus, real-time PCR, calf diarrhea, detection

中图分类号:

S852.659.6

师志海, 王文佳, 兰亚莉, 张彬, 孟红丽, 王亚州, 滑留帅, 徐照学. 牛诺如病毒实时荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2020, 51(7): 1728-1736.

SHI Zhihai, WANG Wenjia, LAN Yali, ZHANG Bin, MENG Hongli, WANG Yazhou, HUA Liushuai, XU Zhaoxue. Establishment and Application of a Real-time PCR Assay for Detecting Bovine Norovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1728-1736.

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牛诺如病毒实时荧光定量PCR检测方法的建立及应用

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

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