新城疫病毒、禽传染性支气管炎病毒与禽流感病毒三重RT-qPCR鉴别检测方法的建立与应用  

Development and Application of a Triple RT-qPCR Assay for the Differential Detection of Newcastle Disease Virus,Avian Infectious Bronchitis Virus and Avian Influenza Virus

作　　者：连聪 向国庆 潘浩华 罗胜军 吕殿红 宋帅 贾春玲 牛瑞辉 郭素茵 李汝洪 邹永基 温肖会 LIAN Cong;XIANG Guoqing;PAN Haohua;LUO Shengjun;LV Dianhong;SONG Shuai;JIA Chunling;NIU Ruihui;GUO Suyin;LI Ruhong;ZOU Yongji;WEN Xiaohui(Zhongkai Agricultural Engineering College,Guangzhou Guangdong 510640;Guangdong Key Laboratory of Livestock Disease Prevention/Guangdong Scientific Observation and ExperimentStation of Veterinary Drugs and Diagnostic Techniques,Ministry of Agriculture and Rural Affairs/Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology/Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Guangzhou Guangdong 510640;Animal Health and Epidemiological Prevention Station,Dongcheng Street,Qingcheng District,Qingyuan Guangdong 511510;Animal Health and Epidemic Prevention Center,Qingcheng District,Qingyuan Guangdong 511599)

机构地区：[1]仲恺农业工程学院,广东广州510225 [2]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站/岭南现代农业科学与技术广东省实验室肇庆分中心,广东广州510640 [3]清远市清城区东城街道动物卫生防疫站,广东清远511510 [4]清远市清城区动物卫生防疫中心,广东清远511599

出　　处：《广东畜牧兽医科技》2024年第3期48-53,共6页Guangdong Journal of Animal and Veterinary Science

基　　金：2022年广东省种畜禽种质资源疫病防控项目;科技创新战略专项资金(高水平农科院建设)(202110TD和R2020PY-JC001);广东省清远市清城区国家现代农业产业园项目;广东省动物疫病野外科学观测研究站项目(2021B1212050021)。

摘　　要：新城疫、禽传染性支气管炎与禽流感是可引起禽类呼吸道症状相似的三种急性传染病,且存在混合感染。为建立新城疫病毒、禽传染性支气管炎病毒与禽流感病毒三重RT-qPCR检测方法,该研究对比了GenBank登录的新城疫病毒(NDV)L基因、禽传染性支气管炎病毒(IBV)1a基因和禽流感病毒(AIV)NP基因,经分析选取保守序列设计了三套引物与探针,通过优化检测方法的反应体系,成功建立了可同时检测NDV、IBV和AIV的三重RT-qPCR方法,并对其特异性、敏感性和重复性进行了评估。结果表明,该方法能够特异性检测出NDV、IBV和AIV,有较好的特异性;方法对三种病毒的最低检测限均为3.02×10^(2)copies/μL,有较高的敏感性;重复性实验结果显示批内与批间变异系数均不大于1.28%,有良好的重复性。使用本研究建立的方法对102份临床咽拭子进行检测,并与常规RT-PCR进行比较,总符合率为96.6%。本研究建立的三重RT-qPCR检测方法,特异性强,灵敏度高,可用于NDV、IBV、AIV三种病毒的检测。Newcastle disease,avian infectious bronchitis and avian influenza are acute infectious diseases that all cause respiratory symptoms in birds,they have similar symptoms and mixed infections may occur.In order to develop a triple fluorescent RT-qPCR method for the detection of NDV,IBV and AI viruses,the present study compared the genes of NDV L gene,IBV 1a gene and AIV NP gene registered in GenBank,and designed three sets of primers and probes by selecting conserved sequences through analysis to optimize the reaction system of the detection method.Ultimately,a triple RT-qPCR method for simultaneous detection of NDV,IBV and AIV was developed successfully,and its specificity,sensitivity and reproducibility were evaluated.The results showed that the method was able to specifically detect NDV,IBV and AIV with good specificity;the lower detection limit of the method was 3.02×10^(2)copies/μL for all the three viruses,indicating a high sensitivity;and the results of the reproducibility experiments showed that the coefficients of variation of intra-and inter-batch were no more than 1.28%,showing an excellent.One hundred and two clinical samples were tested using the method established in this study and the results were compared with those obtained by conventional RT-PCR with an overall agreement rate of 96.6%.The triple RTqPCR assay established in this study is specific and sensitive,and can be used for the detection of the three viruses:NDV,IBV,and AIV.

关 键 词：新城疫病毒 禽传染性支气管炎病毒 禽流感病毒 荧光RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]