DHV-Ⅰ和DEV双重荧光定量PCR方法的建立  

Establishment of DHV-Ⅰand DEV dual fluorescence quantitative PCR method

作　　者：陈肖韩 刘海燕[1] 苏世博 赵丽丽[1] 陈洪岩[1] CHEN Xiao-han;LIU Hai-yan;SU Shi-bo;ZHAO Li-li;CHEN Hong-yan(State Key Laboratory of Veterinary Biotechnology/Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室黑龙江省实验动物与比较医学重点实验室,黑龙江哈尔滨150069

出　　处：《中国兽医科学》2021年第2期169-175,共7页Chinese Veterinary Science

基　　金：国家十三五重点研发计划项目子课题(2016YFD0500800);黑龙江省青年科学基金项目(QC2018033);黑龙江省自然科学基金重点项目(ZD2016006);中央级公益性科研院所基本科研业务费专项(1610302020008,1610302017013)。

摘　　要：为建立快速特异的能够鉴别诊断DHV-I和DEV的荧光定量PCR方法,根据NCBI中I型鸭肝炎病毒和鸭肠炎病毒的保守序列,采用Beacon Designer 7.5软件,设计了2对特异性引物和2条用不同荧光基团标记的TaqMan探针。接着,对反应条件进行优化,验证其特异性及敏感性。结果,该方法特异性好,对鹅细小病毒、番鸭细小病毒、禽流感病毒、新城疫病毒、鸭坦布苏病毒、禽网状内皮增生病毒、禽呼肠弧病毒、减蛋综合征病毒、鸭甲肝病毒3型9种病毒检测,均无荧光信号。而且其灵敏度高,对DHV-I和DEV的最小检出量均为200拷贝。利用所建立的荧光定量PCR方法对江苏徐州采集的166份肛拭子进行检测,检出I型鸭肝炎病毒13份(阳性率为7.8%),鸭肠炎病毒75份(阳性率为45%)。上述结果表明,本研究建立了灵敏度高特异性强的DHV-I和DEV双重荧光定量PCR方法。To establish a rapid and specific fluorescence quantitative PCR method that can differentiate between DHV-I and DEV,according to the conservative sequences of typeⅠduck hepatitis virus and duck enteritis virus in NCBI,using Beacon Designer 7.5 software,two pairs of specific primers and two TaqMan probes labeled with different fluorescent groups were designed.Then optimize the reaction conditions to verify its specificity and sensitivity.In result,the method has good specificity.Avian influenza viruses(AIV),goose parvovirus(GPV),duck Tembusu virus(DTMUV),Newcastle disease virus(NDV),eight viruses including reticuloendotheliosis virus(REV),poultry reovirus(ARV),egg drop syndrome virus(EDSV)and Muscovy duck parvovirus(MDPV)were detected,and no fluorescence signal was found.Moreover,its sensitivity is high,and the minimum detection amount for both DHV-I and DEV is 200 viral genome copies.The established fluorescence quantitative PCR method was used to detect 166 anal swabs collected in Xuzhou,Jiangsu.13 duck hepatitis virus(positive rate 7.8%)and 75 duck plague virus(positive rate 45%)were detected.In conclusion,the DHV-I and DEV dual fluorescence quantitative PCR method with high sensitivity and specificity has been established.

关 键 词：Ⅰ型鸭肝炎病毒 鸭肠炎病毒 双重荧光定量 

分 类 号：S852.659.6[农业科学—基础兽医学]