反刍动物埃立克体荧光定量PCR检测技术的建立和应用

为快速、准确地检测反刍动物埃立克体，本研究以反刍动物埃立克体pCS20为靶基因设计特异性引物和探针，建立了TaqMan和Eva Green荧光定量PCR方法，对其反应的特异性、敏感性和重复性进行了分析，并与OIE推荐的套式PCR方法一起对临床样品进行检测。结果显示，本方法特异性强，与牛巴贝斯虫、牛双芽巴贝斯虫、环形泰勒虫、犬埃立克体、牛埃立克体、马埃立克体和立氏埃立克体无交叉反应；TaqMan和Eva Green荧光定量PCR对pCS20质粒标准品的最低检测限分别为17.4拷贝·μL^-1和1.74拷贝·μL^-1，标准曲线相关系数大于0.99，组内和组间CV均小于1.5%。对420只钝眼蜱样本的检测显示，TaqMan和Eva Green荧光定量PCR的检出率分别为25.48%和29.29%，与套式PCR检测方法相比，敏感性更高。本研究为反刍动物埃立克体的检测和流行病学调查提供了一种快速、准确的检测方法。

Establishment and Application of Real-time PCR Assays for Detection of Ehrlichia ruminantium

In order to detect Ehrlichia ruminantium rapidly and accurately, a set of specific primer and TaqMan probe were designed for TaqMan real-time PCR assay and three sets of specific primers were designed for Eva Green real-time PCR assay according to the pCS20 gene to establish real-time PCR assays for detection of Ehrlichia ruminantium. The specificity, sensitivity and repeatability were tested, respectively. Then the TaqMan and Eva Green real-time PCR were used to detect Amblyomma samples and compared with OIE nested PCR. Results showed that the Ehrlichia ruminantium could be identified specifically and without any cross reaction with B.bovis, B.bigemina, Theileria annulata, E. Canis, E. bovis, E.equi and E. risticii. Besides, the TaqMan and Eva Green real-time PCR detection limits were 17.4 and 1.74 copies·μL^-1, respectively. The coefficients of variations were less than 1.5% for both intra-assay and inter-assay. Total 420 Amblyomma samples were detected by the established assays, positive rate was 25.48% in TaqMan real-time PCR and 29.29% in Eva Green real-time PCR. The sensitivity of TaqMan and Eva Green real-time PCR was significantly higher than that of OIE nested PCR. The results indicated that the detection assays could be applied for rapid and high through-put detection of Ehrlichia ruminantium.