反刍动物埃立克体微滴数字PCR方法的建立及初步应用  

Development and preliminary application of a droplet digital PCR assay for Ehrlichia ruminantium detection

作　　者：王素华 王忠才 黄凌哲 吕继洲[2] 吴绍强[2] 赵治国 付海滨 韩小虎[5] 张舟 WANG Su-hua;WANG Zhong-cai;HUANG Ling-zhe;LV Ji-zhou;WU Shao-qiang;ZHAO Zhi-guo;FU Hai-bin;HAN Xiao-hu;ZHANG Zhou(Comprehensive Technique Service Center of Wenzhou Customs,Wenzhou 325027,China;Institute of Animal Inspection and Quarantine,Chinese Academy of Inspection and Quarantine,Beijing 100029,China;Technology Center of Huhhot Customs,Huhhot 010020,China;Technology Center of Shenyang Customs,Shenyang 110067,China;School of Animal Science and Medicine,Shenyang Agricultural University,Shenyang 110161,China)

机构地区：[1]温州海关综合技术服务中心,浙江温州325027 [2]中国检验检疫科学研究院动物检疫所,北京100029 [3]呼和浩特海关技术中心,内蒙古呼和浩特010020 [4]沈阳海关技术中心,辽宁沈阳110067 [5]沈阳农业大学动物科学与医学学院,辽宁沈阳110161

出　　处：《中国预防兽医学报》2023年第1期60-64,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：浙江省重点研发计划项目(2021C02060);海关总署科技计划项目(2020HK156);中国检验检疫科学研究院基本科研业务费项目(2019JK022)。

摘　　要：为建立反刍动物埃立克体(E.ruminantium)准确定量的微滴数字PCR方法(ddPCR),本研究以E.rum-inantium pCS20基因为靶基因,选取保守区域设计引物和探针,建立了E.ruminantium ddPCR方法。利用本研究建立的ddPCR方法检测E.ruminantium、牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、无形体、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体等,结果显示仅E.ruminantium出现特异性扩增,而与其他寄生虫均无交叉反应,特异性强;将pUC57-pCS20重组质粒标准品10倍倍比稀释(1×10^(-1)拷贝/μL~1×10^(5)拷贝/μL)后,利用该ddPCR方法检测,结果显示,该方法对重组质粒标准品的检测限为1.8拷贝/μL,比相应荧光定量PCR方法的敏感性高10倍,本实验建立的ddPCR方法敏感性高;批内和批间重复性试验结果变异系数(CV)均小于2%,重复性好。利用建立的ddPCR方法对50只钝眼蜱样品检测,结果显示,阳性样品反刍动物埃立克体核酸的最低拷贝数为26拷贝/μL,ddPCR和荧光定量PCR检测试剂盒的检出率均为16.0%(8/50)。本研究首次建立的ddPCR方法敏感性高、特异性强、重复性好,可作为E.ruminantium准确定量检测的有效手段。A droplet digital PCR(ddPCR)assay was established to detect Ehrlichia ruminantium with a pair of specific primers and a TaqMan probe based on the E.ruminantium pCS20 gene.The ddPCR was specific for E.ruminantium,and had no cross-reaction with B.bovis,B.bigemina,Theileria annulata,Anaplasma bovis,E.Canis,E.bovis,E.equi or E.risticii.The detection limit of the ddPCR was 1.8 copies/μL,showing that the ddPCR was 10-fold more sensitive than the real-time PCR previously established in our laboratory.The coefficient of variation of the intra-batch and inter-batch reproducibility tests of the ddPCR were less than 2%.Fifty Amblyomma DNA samples were used to detect E.ruminantium by the established ddPCR and real-time PCR Kit,and positive rates were both 16.0%.All the results showed that the ddPCR detecting E.ruminantium was sensitive and specific.The established ddPCR provides an appropriate detection platform for E.ruminantium.

关 键 词：反刍动物埃立克体 检测 pCS20基因 微滴数字PCR 

分 类 号：S852.61[农业科学—基础兽医学]