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

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.