水貂犬瘟热病毒微滴数字PCR检测方法的建立与应用  

Establishment and application of droplet digital PCR method for detection of canine distemper virus in mink

作　　者：闫曼平 商金源 叶京飞 罗国良[1] 王振军[1] 易立[1] 冯二凯[1] 王春霞 赵宗正 陈明军 魏先力 高华 单晓枫[2] 程悦宁[1] YAN Manping;SHANG Jinyuan;YE Jingfei;LUO Guoliang;WANG Zhenjun;YI Li;FENG Erkai;WANG Chunxia;ZHAO Zongzheng;CHEN Mingjun;WEI Xianli;GAO Hua;SHAN Xiaofeng;CHENG Yuening(Key Laboratory of Economic Animal Diseases,Ministry of Agriculture and Rural Affairs,Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130122,China;College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;Institute of Military Veterinary,Academy of Military Sciences,Changchun 130122,China;Animal Husbandry Station of Xiushui Town,Yushu,Jilin 130436,China;Animal Disease Prevention and Control Center of Horqin District,Tongliao,Inner Mongolia 028000,China)

机构地区：[1]中国农业科学院特产研究所,农业农村部经济动物疫病重点实验室,吉林长春130122 [2]吉林农业大学动物科学技术学院,吉林长春130118 [3]军事科学院军事兽医研究所,吉林长春130112 [4]吉林省榆树市秀水镇畜牧站,吉林榆树130436 [5]通辽市科尔沁区动物疫病预防控制中心,内蒙古通辽028000

出　　处：《中国兽医学报》2023年第12期2445-2450,共6页Chinese Journal of Veterinary Science

基　　金：中国农业科学院科技创新工程资助项目(CAAS-ASTIP-2021-ISAPS);吉林省科技发展计划资助项目(20220508044RC)。

摘　　要：为建立检测水貂犬瘟热病毒(mink canine distemper virus,CDV)的微滴数字PCR(droplet digital PCR,ddPCR)定量方法,以便提供水貂CDV在诊断检测方面的技术支持,本研究根据实时荧光定量PCR(real-time quantitative PCR,qPCR)检测方法原理,建立了水貂CDV数字PCR方法,并优化了反应条件,评估了特异性、敏感性以及重复性。结果表明:ddPCR方法检测CDV的最适引物浓度为900 nmol/L,探针浓度为250 nmol/L,退火温度为55℃,升降温度为2.5℃/s,最低检测下限为4.4拷贝/μL;除CDV特异性扩增,其他常见病毒特异性检测结果均为阴性;重复性试验的变异系数均小于5%。采用该微滴数字PCR和荧光定量RT-PCR方法对30份犬、貂、狐、貉等组织(其中CDV阳性样品7份)样品进行检测,检测结果与临床检测结果相符。本研究建立的微滴数字PCR方法对CDV定量检测特异性强、灵敏度高,重复性好,可以用于水貂CDV临床样品的核酸检测,对水貂CDV发病早期的诊断提供新的定量检测方法。In order to establish droplet digital PCR(ddPCR)for quantitative detection of mink canine distemper virus(CDV),and provide the technical support for diagnosis and detection of mink canine distemper virus(CDV),a digital PCR method for mink distemper virus was established based on the diagnostic principle of real-time quantitative PCR(qPCR).The reaction conditions were optimized and the specificity,sensitivity as well as repeatability were evaluated.The results showed that the optimal concentrations of primers and probes were 900 nmol/L and 250 nmol/L,respectively,with 55℃ for annealing temperature with a 2.5℃/s for up and down-temp erature.The minimum detection limit was 4.4 copies/μL.The specificity assay showed that only CDV was detected as positive,while the other common viruses were all negative,the coefficient of variation in repeatability experiments was less than 5%.Detection of 30 tissue specimens from dogs,minks,foxes and raccoon dogs(including 7 CDV positive samples)by ddPCR and fluorescence quantitative RT-PCR yielded the results consistent with the clinical detection.The ddPCR method established in this study had strong specificity,high sensitivity,and good repeatability for the quantitative detection of CDV,which can be used for the nucleic acid detection among clinical samples of mink distemper virus,providing a new quantitative detection method for early diagnosis of mink distemper virus.

关 键 词：水貂犬瘟热病毒 微滴数字PCR N基因 

分 类 号：S852.65[农业科学—基础兽医学]