ALV-J、REV和MDV多重荧光定量PCR检测方法的建立  

ESTABLISHMENT OF A TRIPLE REAL-TIME PCR FOR DETECTING ALV-J,REV AND MDV

作　　者：汪最 李丽 刘鹏 刘丽娜[1,2] 汪琛 周康 罗青平 WANG Zui;LI Li;LIU Peng;LIU Li-na;WANG Chen;ZHOU Kang;LUO Qing-ping(Llnstitute of Animal Husbandry and Veterinary,Hubei Academy of Agricultural Sciences,Wuhan 430064,China;Key Laboratory of Animal Disease Control and Prevention Formulation of Ministry of Agriculture and Rural Affairs,Wuhan 430064,China;Xuanen County Animal Husbandry and Veternary Bureau,Enshi 445500,China)

机构地区：[1]湖北省农业科学院畜牧兽医研究所,武汉430064 [2]农业农村部畜禽细菌病防治制剂创制重点实验室,武汉430064 [3]宣恩县畜牧兽医局,恩施445500

出　　处：《中国动物传染病学报》2020年第6期73-78,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划(2015YFD0500803);现代农业产业技术体系建设专项资金(CARS-41-G13);湖北省畜禽优质增效与绿色养殖科技创新行动(2018skjcx05)

摘　　要：为建立可同时检测J亚群禽白血病病毒(ALV)、网状内皮增生症病毒(REV)和马立克病病毒(MDV)的方法,本研究以ALV-J的ENV基因、REV的LTR区以及MDV的MEQ基因为检测对象,建立了可同时检测ALV-J、REV、MDV的多重荧光定量PCR方法并对其特异性、灵敏性、稳定性进行了研究。结果显示,该方法仅可特异扩增3种肿瘤病病毒的基因,对禽流感病毒、新城疫病毒、腺病毒等核酸扩增均呈阴性;ALV-J、REV和MDV的最低核酸检测限分别为35 copies/μL、5.1 copies/μL和62 copies/μL;此外,批内与批间重复试验Ct值的变异系数均小于2%。利用此方法对166份送检样品进行检测,其中ALV-J、REV和MDV检出率分别为25.3%、7.2%和5.4%,混合感染检出率为3.6%。以上结果表明,本方法特异性好、灵敏度高、稳定性强,能够同时检测ALV-J、REV和MDV,可用于禽肿瘤病的鉴别诊断、流行病学调查以及疫病防控。In order to develop a method for simultaneous detection of Avian leukosis virus (ALV),Reticuloendotheliosis virus (REV) and Marek’s disease virus (MDV),a triple real-time PCR was established using primers specific for ENV of ALV-J,LTR of REV and MEQ of MDV and its specificity,sensitivity and stability were studied subsequently.The results showed that this method could specific for detecting ALV-J,MDV and REV with the detection limit of 35 copies/μL,5.1 copies/μL and 62 copies/μL,respectively and had not any amplification for AIV,NDV,FAdV and other viruses.In addition,the variation coefficients of repeated tests within and between batches were both less than 2%.Furthermore,166 tissue samples of chicken were detected using this method and the positive rates of ALV-J,REV and MDV were 25.3%,7.2% and 5.4%,respectively,and the rate of mixed infection was 3.6%.The above results showed this method had the characteristics of good specificity,high sensitivity and strong stability,and can simultaneously detect ALV-J,REV and MDV,which can be used for the differential diagnosis,epidemiological investigation and epidemic prevention and control of avian cancer diseases.

关 键 词：禽白血病病毒 网状内皮增生症病毒 马立克病病毒 多重荧光定量PCR 

分 类 号：S852.659.3[农业科学—基础兽医学]