鉴别感染羊驼4种病毒多重PCR检测方法的建立  

Development of a multiplex PCR assay for identification of 4 viruses in alpaca

作　　者：于远光[1] 岳涛 马晓宁 张永兴[1] 王萌[2] 曾巧英[2] 刘艳红[1] 高闪电[1] 独军政[1] 吴锦艳[1] 李坤[1] 许小红[3] YU Yuan-guang;YUE Tao;MA Xiao-ning;ZHANG Yong-xing;WANG Meng;ZENG Qiao-ying;LIU Yan-hong;GAO Shan-dian;DU Jun-zheng;WU Jin-yan;LI Kun;XU Xiao-hong(State Key Laboratory for Animal Disease Control and Prevention/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Gansu Agricultural University,Lanzhou 730070,China;Gansu Provincial Center for Disease Control and Prevention,Lanzhou 730000,China)

机构地区：[1]中国农业科学院,兰州兽医研究所,动物疫病防控全国重点实验室,甘肃兰州730046 [2]甘肃农业大学,甘肃兰州730070 [3]甘肃省疾病预防控制中心,甘肃兰州730000

出　　处：《中国兽医科学》2023年第9期1102-1107,共6页Chinese Veterinary Science

基　　金：中央引导地方科技发展资金项目(YDZX20216200004349);甘肃省科技计划项目(22JR5RA715)。

摘　　要：为了建立一种用于羊驼易感的羊口疮病毒(ORFV)、牛病毒性腹泻病毒(BVDV-1)、口蹄疫病毒(FMDV)、蓝舌病病毒(BTV)鉴别的多重PCR检测方法,根据GenBank中公布的ORFV、BVDV-1、FMDV和BTV参考株基因序列分别设计特异性引物,优化引物、dNTP、Taq DNA聚合酶浓度及退火温度,通过敏感性和重复性检验,并对60份羊驼粪便样品和35份羊驼咽拭子样品进行验证。结果显示,所建立的多重PCR方法能够有效区分ORFV、BVDV-1、FMDV和BTV,最低检测限分别为3.18×10^(4)copies/μL、3.13×10^(1)copies/μL、3.06×10^(1)copies/μL、2.95×10^(2)copies/μL。结论:所建立的多重PCR方法能够同时快速鉴别羊驼感染的ORFV、BVDV-1、FMDV和BTV,具有较高的应用价值。To establish a multiplex polymerase chain reaction(multi-PCR)assay for identification of sheep mouth sore virus(ORFV),bovine viral diarrhea virus(BVDV-1),foot-and-mouth disease virus(FMDV),bluetongue virus(BTV)in alpaca,4 specific primers were designed according to sequences of the four pathogens from GenBank,respectively,and the multi-PCR assay was optimized for the concentration of primers,dNTP and Taq DNA polymerase,the annealing temperature,the sensitivity and reproducibility were verified,60 stool samples and 35 throat swab samples taken from laboratory alpacas were assayed.The results indicated that this multi-PCR method can distinguish ORFV,BVDV-1,FMDV and BTV,the sensibility was 3.18×10^(4) copies/μL ORFV,3.13×10^(1) copies/μL BVDV-1,3.06×10^(1) copies/μL FMDV and 2.95×10^(2) copies/μL BTV,respectively.In conclusion,the multi-PCR assay developed in this study can efficiently identificate ORFV,BVDV-1,FMDV and BTV,representing a useful technique for rapid detection of the viruses.

关 键 词：羊驼 口蹄疫病毒 羊口疮病毒 蓝舌病病毒 牛病毒性腹泻病毒 多重PCR 

分 类 号：S852.65[农业科学—基础兽医学]