检测牛5种腹泻病毒的多重RT-PCR方法的建立及应用  

Establishment and Application of Multiplex RT-PCR for Detection of Five Bovine Diarrhea Viruses

作　　者：孙吉 岳华[1,2] 汤承 SUN Ji;YUE Hua;TANG Cheng(Institute of Animal Husbandry and Veterinary Medicine,Southwest Minzu University,Chengdu 610041,China;Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization,Chengdu 610041,China)

机构地区：[1]西南民族大学畜牧兽医学院,成都610041 [2]青藏高原动物遗传资源保护与利用教育部重点实验室,成都610041

出　　处：《畜牧兽医学报》2022年第1期209-218,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家农业产业技术体系四川肉牛创新团队专项(SCCXTD-2020-13);西南民族大学新发动物疫病研究创新团队(2020NTD02);西南民族大学动物医学实验室平台建设项目(2020PTJS29)。

摘　　要：牛A群轮状病毒(BRVA)、牛冠状病毒(BCoV)、牛病毒性腹泻病毒(BVDV)、牛诺瓦病毒(BNoV)和牛纽布病毒(BNeV)是引起犊牛腹泻的常见病毒,且临床上多存在混合感染的情况。本试验的目的是建立可以同时检测上述5种病毒的多重PCR方法。通过设计引物,优化反应体系和条件,成功建立了可同时检测BRVA、BCoV、BVDV、BNoV和BNeV的多重PCR方法。该方法只特异性检出目标病毒,而对牛星状病毒、牛嵴病毒、牛细小病毒、产肠毒素大肠杆菌、沙门菌和空肠弯曲杆菌等腹泻病原均无扩增,具有良好的特异性和稳定性。对BRVA、BCoV、BVDV、BNoV、BNeV的最低检测下限分别为1.63×10^(5)、2.0×10^(6)、1.9×10^(5)、1.96×10^(5)和2.0×10^(5)copies·μL^(-1)。比较试验表明,该多重RT-PCR方法与检测单一病毒的RT-PCR方法的符合率为100%。利用该方法对2019—2020年采集自川西北草原的220份牦牛腹泻样本的检测结果表明,BRV的检出率为40.5%、BNoV的检出率为5.9%、BNeV的检出率为4.5%,而BVDV和BCoV无检出。本研究建立的多重RT-PCR方法可同时检测犊牛腹泻粪便样品中的5种常见腹泻病毒,为牛腹泻的快速诊断提供了技术支持。Bovine group A rotavirus(BRVA),bovine coronavirus(BCoV),bovine viral diarrhea virus(BVDV),bovine norovirus(BNoV)and bovine nebovirus(BNeV)are common viruses that cause calf diarrhea,and there are mixed infections in clinic.The purpose of this experiment was to establish a multiplex PCR method that can simultaneously detect the above five viruses.The multiplex PCR method for simultaneous detection of BRVA,BCoV,BVDV,BNoV and BNeV was successfully established by designing primers,optimizing reaction system and conditions.This method only specifically detected the target virus,but did not amplify the diarrhea pathogens such as bovine astrovirus,bovine kobuvirus,bovine parvovirus,enterotoxigenic Escherichia coli,Salmonella and Campylobacter jejuni,which had good specificity and stability.The lowest detection limits for each virus were as follows:BRVA 1.63×10^(5)copies·μL^(-1),BCoV 2.0×10^(6) copies·μL^(-1),BVDV 1.9×10^(5) copies·μL^(-1),BNoV 1.96×10^(5) copies·μL^(-1) and BNeV 2.0×10^(5) copies·μL^(-1).The comparison experiment showed that the coincidence rate between the multiplex RT-PCR method and the RT-PCR method for detecting single virus was 100%.The detection results of 220 yak diarrhea samples collected from northwest Sichuan grassland from 2019 to 2020 showed that the detection rates of BRVA,BNoV and BNeV were 40.5%,5.9%and 4.5%,respectively,while BVDV and BCoV were not detected.The multiplex RT-PCR method established in this study can simultaneously detect five common diarrhea viruses in fecal samples of calf diarrhea,providing technical support for the rapid diagnosis of bovine diarrhea.

关 键 词：牛A群轮状病毒 牛冠状病毒 牛病毒性腹泻病毒 牛诺瓦病毒 牛纽布病毒 多重RT-PCR 

分 类 号：S855.3[农业科学—临床兽医学]