环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立

中国畜牧兽医

环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立

郑进峰^1,2，罗金²，谢俊仁²，田美媛²，袁小松²，王芳芳²，郭锦霞¹，李奎¹，刘光远²

（1.河南农业大学牧医工程学院，河南郑州 450002；2. 中国农业科学院兰州兽医研究所，家畜疫原病原生物学国家重点实验室，甘肃省动物寄生虫病重点实验室，甘肃兰州 730046）

出版日期:2014-01-20 发布日期:2014-02-27
通讯作者: 李奎（1962—），男，教授，研究方向：动物解剖与组织学。E-mail:likui0420@sina.com 刘光远（1963—），男，研究员，研究方向：动物寄生虫学。E-mail:liuguangyuan2002@sina.com
作者简介:郑进峰（1989—），男，河南人，硕士生，研究方向：牛羊寄生虫免疫。
基金资助:
国家自然科学基金项目（31201899）。

stablishment of PCR Method for the Detection of Theirelia annulata Based on TaSP Gene

ZHENG Jin-feng^1,2, LUO Jin², XIE Jun-ren², TIAN Mei-yuan²,YUAN Xiao-song²,WANG Fang-fang², GUO Jin-xia¹, LI Kui¹，LIU Guang-yuan²

450002,China；2. Key Laboratory of Veterinary Parasitology of Gansu Province，Key Laboratory of Grazing Animal Diseases of Ministry of Agriculture，State Key Laboratory of Veterinary Etiological Biology，Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences, Lanzhou 730046,China)

Online:2014-01-20 Published:2014-02-27

摘要/Abstract

摘要： 为寻求一种快速灵敏的环形泰勒虫病PCR检测方法，基于环形泰勒虫裂殖体表面蛋白（Theirelia annulata surface protein,TaSP）基因序列保守区设计特异性引物，通过PCR技术扩增出该基因长为393 bp的高免疫原性区片段。用该引物对环形泰勒虫、中华泰勒虫、瑟式泰勒虫、尤氏泰勒虫、吕氏泰勒虫、绵羊泰勒虫、马泰勒虫、驽巴贝斯虫、牛巴贝斯虫基因组模板进行特异性试验，对环形泰勒虫基因组模板进行梯度稀释后扩增，以确定试验的敏感性，同时用本试验建立的方法与常规显微镜镜检方法对150份血清样品进行检测。特异性试验结果显示，在被检测的9个样本中，只有环形泰勒虫基因组模板中扩增出了符合大小的特异核苷酸片段；敏感性试验结果表明，PCR对环形泰勒虫的扩增效率可达到10^-10；通过对150份血清样品的检测，并与血涂片方法进行比较，结果显示PCR方法具有特异性强、敏感度高等特点，适用于牛环形泰勒虫病的检测。

关键词: 环形泰勒虫; 环形泰勒虫裂殖体表面蛋白（TaSP）基因; PCR诊断方法

Abstract: To establish a PCR method of Theirelia annulata （T. annulata）, a pair of primers was designed to specifically amplify a 393 bp high immunization fragment based on T. annulata surface protein （TaSP） gene conserved region，T. annulata, T. sinensis, T. sergenti, T. uilenbergi, T. luwenshuni, T. ovis, T. equi, B. caballi and B. bovis were tested by PCR with the primers. While T. annulata genome templates were amplified that different concentrations diluted in order to determine the sensitivity of the experiment. 150 bovine blood samples were detected using PCR and microscopic. The PCR results of specificity assay showed that only T. annulata genome template amplified specific fragment. The sensitivity result showed that the minimum dose of T. annulata that could be detected by PCR assay was 10^-10. 150 blood samples were detected by PCR assay and microscopic examination of Giemsa-stained blood semars,the results showed that PCR assay was sensitive and specific. It was suitable for the detection of T. annulata.

Key words: Theirelia annulata; TaSP gene; PCR method

郑进峰，罗金，谢俊仁，田美媛，袁小松，王芳芳，郭锦霞，李奎，刘光远. 环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立[J]. 中国畜牧兽医.

ZHENG Jin-feng, LUO Jin, XIE Jun-ren, TIAN Mei-yuan,YUAN Xiao-song,WANG Fang-fang, GUO Jin-xia, LI Kui，LIU Guang-yuan. stablishment of PCR Method for the Detection of Theirelia annulata Based on TaSP Gene[J]. China Animal Husbandry & Veterinary Medicine.

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