貂肠炎病毒微滴数字PCR检测方法的建立与应用  

Establishment and application of droplet digital PCR assay for quantitative detection of mink enteritis virus

作　　者：商金源 叶京飞 罗国良[1] 王振军[1] 冯二凯[1] 王春霞 贾北宁 李斌茹 赵宗正 陈明军 魏先力 程悦宁[1] SHANG Jin-yuan;YE Jing-fei;LUO Guo-liang;WANG Zhen-jun;FENG Er-kai;WANG Chun-xia;JIA Bei-ning;LI Bin-ru;ZHAO Zong-zheng;CHEN Mingjun;WEI Xian-li;CHENG Yue-ning(Key Laboratory of Economic Animal Diseases,Ministry of Agriculture/lnstitute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130122,China;Agricultural Development Service Center of Nanpiao District,Huludao 125027,China;Liutaizhuang Animal Husbandry and Veterinary Center Station,Qinhuangdao 066600,China;Institute of Military Veterinary,Academy of Military Sciences,Changchun 130122,China;Animal Husbandry Station of Xiushui Town,Yushu130436,China)

机构地区：[1]中国农业科学院特产研究所农业部经济动物疫病重点实验室,吉林长春130122 [2]辽宁省葫芦岛市南票区农业发展服务中心,辽宁葫芦岛125027 [3]刘台庄畜牧兽医中心站,河北秦皇岛066600 [4]军事科学院军事兽医研究所,吉林长春130112 [5]吉林省榆树市秀水镇畜牧站,吉林榆树130436

出　　处：《中国兽医科学》2023年第2期156-162,共7页Chinese Veterinary Science

基　　金：吉林省科技发展计划项目(20200402110NC,20200402036NC);2022年吉林省发改委研究课题(2022C042-9);中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-ISAPS)。

摘　　要：建立一种貂肠炎病毒(MEV)微滴数字PCR(ddPCR)方法,对貂肠炎病毒的定量诊断提供技术支持。在实时荧光定量PCR(qPCR)检测方法的基础上,建立了貂肠炎病毒微滴数字PCR方法,优化了该检测方法的反应条件,并评估了其敏感性、特异性、重复性。结果显示,建立的ddPCR方法最佳引物和探针终浓度分别为900和250 nmol/L,最佳退火温度为55℃,最佳升降温速度为2.5℃/s,本方法的最低检测下限为1.07×10^(0)copies/μL,未发现与常见疫病病毒存在交叉反应,重复性试验的变异系数小于5%。采用该微滴数字PCR和实时荧光PCR对40份水貂肠道、粪便样品进行检测,检测结果与临床检验结果相符。结果表明,本研究中建立的微滴数字PCR方法敏感性高、特异性强、重复性好,适用于临床样品的貂肠炎病毒核酸检测,为貂肠炎病毒感染的早期检测提供了技术支持。To investigate the use of droplet digital polymerase chain reaction(ddPCR)method for detecting mink enteritis virus(MEV)and providing technical support for quantitative diagnosis of MEV,the dd PCR method for MEV was established on the basis of real-time fluorescence PCR detection method,and the reaction conditions of detection method were optimized.The results showed that the best final concentration of primers and probe were 900 nmol/L and 250 nmol/L,respectively,the best annealing temperature was 55℃,and the best temperature rate of change was 2.5℃/s.The results showed that the minimum detection limit of this method was 1.07×10^(0) copies/μL,and the coefficient of variation of repeatability test was less than 5%.The dd PCR and fluorescence real-time PCR were used to detect 40 samples of mink intestinal and fecal,and the detection results were consistent with the clinical test results.The dd PCR method established in this study has high sensitivity,high specificity and good repeatability.It is suitable for nucleic acid detection of MEV in clinical samples and can be used in early detection of MEV infection.

关 键 词：貂肠炎病毒 微滴数字PCR 定量检测 

分 类 号：S852.659.2[农业科学—基础兽医学]