反刍动物埃立克体荧光定量PCR检测技术的建立和应用

doi:10.11843/j.issn.0366-6964.2021.07.019

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (7): 1975-1982.doi: 10.11843/j.issn.0366-6964.2021.07.019

反刍动物埃立克体荧光定量PCR检测技术的建立和应用

王素华^1*, 王忠才¹, 黄凌哲¹, 莫虹斐², 吴绍强³, 吕继洲³, 赵治国⁴, 帅江冰²

1. 温州海关综合技术服务中心, 温州 325027;
2. 杭州海关技术中心, 杭州 310012;
3. 中国检验检疫科学研究院动物检疫所, 北京 100029;
4. 呼和浩特海关技术中心, 呼和浩特 010020

收稿日期:2020-11-27 出版日期:2021-07-23 发布日期:2021-07-23
通讯作者: 王素华,E-mail:29801719@qq.com
作者简介:王素华(1975-),女,山东聊城人,高级兽医师,硕士,主要从事动物及动物产品检疫研究
基金资助:
浙江省自然科学基金项目（LGN19C180001）；海关总署科技计划项目（2020HK156）

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

WANG Suhua^1*, WANG Zhongcai¹, HUANG Lingzhe¹, MO Hongfei², WU Shaoqiang³, Lü Jizhou³, ZHAO Zhiguo⁴, SHUAI Jiangbing²

1. Synthesis Technique Service Center of Wenzhou Customs, Wenzhou 325027, China;
2. Technology Center of Hangzhou Customs, Hangzhou 310012, China;
3. Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China;
4. Technology Center of Hohhot Customs, Hohhot 010020, China

Received:2020-11-27 Online:2021-07-23 Published:2021-07-23

摘要/Abstract

摘要： 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。

关键词: 反刍动物埃立克体, pCS20基因, TaqMan荧光定量PCR, Eva Green荧光定量PCR

Abstract: 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.

Key words: Ehrlichia ruminantium, pCS20 gene, TaqMan real-time PCR, Eva Green real-time PCR

中图分类号:

S852.61

王素华, 王忠才, 黄凌哲, 莫虹斐, 吴绍强, 吕继洲, 赵治国, 帅江冰. 反刍动物埃立克体荧光定量PCR检测技术的建立和应用[J]. 畜牧兽医学报, 2021, 52(7): 1975-1982.

WANG Suhua, WANG Zhongcai, HUANG Lingzhe, MO Hongfei, WU Shaoqiang, Lü Jizhou, ZHAO Zhiguo, SHUAI Jiangbing. Establishment and Application of Real-time PCR Assays for Detection of Ehrlichia ruminantium[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1975-1982.

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反刍动物埃立克体荧光定量PCR检测技术的建立和应用

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

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