犬瘟热病毒(CDV) RPA-SYBR Green Ⅰ 检测方法的建立与应用  

Rapid detection of canine distemper virus by recombinase polymerase amplification combined with RPA-SYBR Green Ⅰ method

作　　者：郝镯 张珊珊 李楠 田多 李吉平 孟轲音 万忠海 穆玉堂 李忠义 王承宇 HAO Zhuo;ZHANG Shanshan;LI Nan;TIAN Duo;LI Jiping;MENG Keyin;WAN Zhonghai;MU Yutang;LI Zhongyi;WANG Chengyu(Key Laboratory of Jilin Province for Zoonosis Prevention and Control,Institute of Military Veterinary Science,The Academy of Military Medical Science,Academy of Military Science,Changchun 130122,China)

机构地区：[1]军事科学院军事医学研究院军事兽医研究所/吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国兽医学报》2021年第5期905-910,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0501001)。

摘　　要：为建立一种新型、稳定、可普遍应用的诊断犬瘟热病毒(CDV)的方法,将重组酶聚合酶扩增(recombinase polymerase amplification, RPA)与SYBR GreenⅠ相结合,根据CDV N基因序列中同源性相对较高的保守区设计1套标有生物素Biotin的特异性引物和1条标记dSpacer、C3 Spacer的特异性探针,优化反应条件和体系,分析其灵敏性及特异性,最后建立一种可以进行临床应用的,特异性检测CDV的RPA-SYBR GreenⅠ方法。结果显示,成功构建检测CDV的RPA-SYBR GreenⅠ方法,此方法的最佳反应条件为实验人员闭合手掌中孵育10 min,引物与探针的最佳比例为2∶1。该方法的最低检测下限为1×10^(1)~1×10^(0) TCID_(50)/mL,能够特异地检测出CDV。应用本方法检测临床样品的结果与ELISA、RT-PCR方法的结果一致。结果表明,本试验所建立的CDV RPA-SYBR GreenⅠ检测方法特异性强,灵敏度高,操作简单,可应用于临床。In order to establish a new, stable and universally applicable method for the diagnosis of canine distemper virus(CDV),recombinase polymerase amplification(RPA)-SYBR Green Ⅰ technique was established.According to the conserved region with relatively high homology in the N gene sequence of CDV,a set of specific primers labeled with Biotin was designed, and a specific probe for labeling dSpacer and C3 Spacer was combined to establish RPA-SYBR Green Ⅰ detection method.The results showed that the CDV RPA-SYBR Green Ⅰ method was successfully established, and the optimum reaction conditions were in the palm of the hand for 10 min could initiate RPA amplification reaction.The optimal ratio of primers to probes was 2∶1,and the minimum detection limit was between the 1×10^(1)-1×10^(0) TCID_(50)/mL.CDV could be specifically detected, and other viruses such as canine parvovirus(CPV) were all negative.It showed that the positive rate of RPA-SYBR Green Ⅰ method in clinical samples were consistent with that of ELISA and RT-PCR methods.These results indicated that the established method of CDV RPA-SYBR Green Ⅰ had strong specificity, high sensitivity, simple operation and could be widely used.

关 键 词：CDV RPA SYBR GreenⅠ N基因 重组酶聚合酶扩增 

分 类 号：S855.3[农业科学—临床兽医学]