禽呼肠孤病毒纳米PCR检测方法的建立与应用  

Establishment and application of nano PCR assay for detection of avian reovirus

作　　者：郑丽[1,2] 任卫科 路超[1,2] 田向学 王利丽[1,2] 李秀丽 鄢明华[1,2] ZHENG Li;REN Wei-ke;LU Chao;TIAN Xiang-xue;WANG Li-li;LI Xiu-li;YAN Ming-hua(Tianjin Institute ofA nima! Husbandry and Veterinary Science, Tianjin 300381, China;Tianjin EngineeringResearch Center for Livestock and Poultry Health Breeding, Tianjin 300381, China)

机构地区：[1]天津市畜牧兽医研究所,天津300381 [2]天津市畜禽健康养殖技术工程中心,天津300381

出　　处：《中国兽医科学》2018年第6期700-706,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2016YFD0500800)

摘　　要：为建立一种快速、敏感的禽呼肠孤病毒（ARV）检测方法,针对ARV S3基因的保守区域设计1对特异性引物,建立了ARV纳米PCR检测方法。结果显示,该方法特异性好,仅从ARV核酸样品可检测到约332 bp的特异性目的条带,而对新城疫病毒、传染性支气管炎病毒、传染性囊病病毒、鸡毒支原体、马立克病病毒、传染性喉气管炎病毒、鸭瘟病毒、禽腺病毒Ⅳ型、减蛋综合征病毒、H9亚型禽流感病毒和鸡大肠杆菌等其他病原的检测结果均为阴性。敏感性试验结果显示,该纳米PCR方法能检测到的最低核酸质量浓度为56 pg/L,其灵敏度是普通PCR的10倍。ARV核酸被重复检测3次,结果均一致。应用建立的纳米PCR和病毒分离鉴定方法对临床送检的30份样品进行检测,两种方法的符合率为100%。以上结果表明,成功建立了ARV的纳米PCR检测方法,具有更高的敏感性和良好的特异性,可用于ARV感染的临床诊断及流行病学调查。In order to develop a rapid and sensitive method for detecting avian reovirus（ARV）,a pair of primers was designed based on the conserved region of ARV S3 gene,and an ARV nano PCR assay was established.The results showed only ARV was successfully amplified with a target fragment of 332 bp in size,but the amplification results were negative for Newcastle disease virus,infectious bronchitis virus,infectious bursal disease virus,Mycoplasma gallisepticum,Marek＇s disease virus,infectious laryngotracheitis virus,duck plaque virus,fowl adenovirus type 4,egg drop syndrome virus,avian influenza virus（H9） and Escherichia coli.The sensitivity test showed that the sensitivity of the nano PCR was 10 times as high as that of the conventional PCR,and the lowest detection mass concentration of c DNA was 56 pg/L.ARV nucleic acid was detected 3 times by the nano PCR,and these results were consistent.Furthermore,30 clinical samples were detected by the nano PCR and virus isolation methods,and the coincidence rate of the two methods was 100%.The above results showed that the ARV nano PCR method was successfully established,which has high sensitivity and good specificity,and the method can be used for clinical diagnosis and epidemiological investigation of ARV infection.

关 键 词：禽呼肠孤病毒 S3基因 纳米PCR 

分 类 号：S852.659.4[农业科学—基础兽医学]