鸡星状病毒、禽肾炎病毒和鸡传染性支气管炎病毒多重PCR检测方法的建立  

Development of a Multiplex PCR to Discriminate Chicken Astrovirus,Avian Nephritis Virus and Infectious Bronchitis Virus

作　　者：栾庆东 曹旭 尹燕博[1] 王建琳[1] LUAN Qing-dong;CAO Xu;YIN Yan-bo;WANG Jian-lin(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医杂志》2022年第3期32-36,41,共6页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划项目(2016YFD0500800);山东省现代农业产业技术体系项目(SDAIT-11-03)。

摘　　要：为有效确定引起鸡痛风的病原,本试验建立了同时检测鸡星状病毒(CAstV)、禽肾炎病毒(ANV)和鸡传染性支气管炎病毒(IBV)的多重PCR方法。针对IBV的N基因、CAstV和ANV的ORF-1b基因分别设计了3对特异性引物,通过优化退火温度等反应条件建立了多重PCR检测方法,并评估了该方法的特异性和敏感性,而后对临床痛风样本进行了检测。结果显示,多重PCR方法对3种病毒扩增产物的大小分别为1 600 bp(IBV)、794 bp(ANV)和350 bp(CAstV);该多重PCR检测禽马立克氏病毒、血清4型禽腺病毒、减蛋综合征病毒和J亚型禽白血病病毒均为阴性;病毒最低检测限IBV为1.96×10^(2) copies/μL,ANV为2.10×10^(2) copies/μL,CAstV为1.33×10^(5)copies/μL;多重PCR对临床病料的检测结果与单项PCR的检测结果一致。结果表明,本试验建立的多重PCR检测方法具有较好的特异性、敏感性和可靠性,为临床鸡痛风的诊断提供了准确、有效的技术手段。In order to identify the viruses that cause gout in chicken,this study developed a multiplex PCR for simultaneous detection of chicken astrovirus(CAstV),avian nephritis virus(ANV) and avian infectious bronchitis virus(IBV).Three pairs of primers were designed from the N gene of IBV,the ORF-1b gene of CAstV and ANV,evaluated in multiplex PCR in relation to their specificity and sensitivity,and further assessed with clinical gout samples.The results showed that the multiplex PCR generated three specific bands of about 1 600 bp(IBV),794 bp(ANV)and 350 bp(CAstV)from the nucleic acids of IBV,ANV and CAstV,respectively,but not of Marek′s disease virus,avian leukosis virus,fowl adenovirus-4 and egg drop syndrome virus.The multiplex PCR had detection limits of 1.96×10~2,2.10×10~2 copies/μL and 1.33×10^(5 )copies/μL of IBV,ANV and CAstV,respectively,and performed similarly to single PCR with clinical gout samples.Thus,the newly developed multiplex PCR has good specificity,sensitivity and reliability,and offers a specific and sensitive approach for simultaneous detection of IBV,ANV and CAstV from clinical gout samples.

关 键 词：鸡痛风 鸡星状病毒 禽肾炎病毒 鸡传染性支气管炎病毒 多重PCR 

分 类 号：S855.3[农业科学—临床兽医学]