基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立

中国寄生虫学与寄生虫病杂志 ›› 2017, Vol. 35 ›› Issue (2): 169-172.

基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立

张艳艳¹, 叶倩², 王正荣¹, 薄新文^1,^*()

1新疆农垦科学院,省部共建绵羊遗传改良与健康养殖国家重点实验室,石河子 832000
2 石河子大学,石河子 832000

收稿日期:2016-06-07 出版日期:2017-04-20 发布日期:2017-05-02
通讯作者: 薄新文
基金资助:
新疆生产建设兵团科技创新团队(No. 2014CC004);新疆生产建设兵团科技攻关项目(No. 2014BA002);新疆生产建设兵团国际科技合作项目(No. 2013BC001)

Preliminary exploration of loop-mediated isothermal amplification based on cox2 gene of Echinococcus granulosus

Yan-yan ZHANG¹, Qian YE², Zheng-rong WANG¹, Xin-wen BO^1,^*()

1 State Key Laboratory for Sheep Genetic Improvement and Healthy Production/Xinjiang Academy of Agricultural and Reclamation Science, Shihezi 832000, China
2 College of Animal Science and Technology, Shihezi University, Shihezi 832000, China

Received:2016-06-07 Online:2017-04-20 Published:2017-05-02
Contact: Xin-wen BO
Supported by:
Supported by the Science and Technology Innovation Team in Xinjiang Production and Construction Corps(No. 2014CC004), Science and Technique Fund of Xinjiang Production and Construction Corps(No. 2014BA002), and International Science and Technology Cooperation Project of Xinjiang Production and Construction Corps(No. 2013BC001)

摘要/Abstract

摘要：

目的建立一种安全、快速、高效的细粒棘球绦虫（Echinococcus granulosus）的诊断方法——环介导等温扩增方法(loop-mediated isothermal amplification,LAMP)。方法根据细粒棘球绦虫线粒体细胞色素c氧化酶亚基2（cytochrome c oxidase subunit 2,cox2）基因序列,设计4条LAMP引物,建立LAMP检测方法,采用LAMP法和常规PCR法检测细粒棘球绦虫、泡状带绦虫(Cysticercus tenuicollis )、扩展莫尼茨绦虫（Moniezia expansa）、羊曲子宫绦虫(Thysaniezia ovilla)、中点无卵黄腺绦虫（Avitellina centripunctata）、鞭毛线虫（flagella nematodes）、肝片吸虫（Fasciola hepatica）、多房棘球绦虫（E. multilocularis）和捻转血矛线虫（Haemonchus contortus）等9种寄生虫的DNA以验证LAMP法的特异性;分别用LAMP法和常规PCR法检测梯度稀释的含cox2基因片段的细粒棘球绦虫标准质粒后,比较两者的敏感性。采用LAMP、PCR和夹心ELISA法检测50份犬粪样,计算细粒棘球绦虫cox2基因的阳性率,评价所建立的LAMP法检测效果。结果 LAMP法的特异性验证结果表明,仅在以细粒棘球绦虫DNA为模板的反应体系中出现特异性产物。LAMP法在细粒棘球绦虫标准质粒浓度为16.09 ag/μl时仍可见梯状条带,而常规PCR法仅可检测到浓度为16.9 fg/μl以上的质粒,LAMP法灵敏性是常规PCR法的1 000倍。对50份犬粪中细粒棘球绦虫DNA的检测结果显示,PCR、LAMP和夹心ELISA法检测结果相同,阳性率为8.0%（4/50）。结论基于细粒棘球绦虫线粒体cox2基因的LAMP检测方法操作方便、特异性较强、敏感性较高,适于细粒棘球绦虫感染犬的快速检测。

关键词: 细粒棘球绦虫, 细胞色素c氧化酶亚基2基因, 环介导等温扩增

Abstract:

Objective To establish a loop-mediated isothermal amplification(LAMP) assay for rapid detection of Echinococcus granulosus cytochrome c oxidase subunit 2(cox2) gene in dog feces. Methods Four primers were designed based on the cox2 gene of Echinococcus granulosus and were used for the LAMP assay. LAMP assay and conventional PCR were performed to detect E. granulosus, Cysticercus tenuicollis, Moniezia expansa, Thysaniezia ovilla, Avitellina centripunctata, flagella nematode, Fasciola hepatica, E. multilocularis and Haemonchus contortus, and the specificity of LAMP was evaluated. The sensitivity of LAMP was compared with that of conventional PCR using gradient dilutions of recombinant plasmid carrying cox2 fragment of E. granulosus. In addition, LAMP, conventional PCR and ELISA were performed to detect E. granulosus in 50 dog fecal samples. The positive rate of E. granulosus was calculated and the efficacy of LAMP was evaluated. Results The LAMP assay only produced specific bands for E. granulosus among various species. The sensitivity of LAMP for E. granulosus was 16.09 ag/μl, which was 1 000 times higher than that of conventional PCR(16.9 fg/μl). Regarding the fecal samples, the three methods revealed same positive rate of 8.0%(4/50). Conclusion The LAMP assay is a convenient method to detect E. granulosus based on the cox2 gene with a high specificity and sensitivity, revealing a suitable technique for rapid detection of E. granulosus in dogs.

Key words: Echinococcus granulosus, Cytochrome c oxidase subunit 2, LAMP

中图分类号:

R383.21

张艳艳, 叶倩, 王正荣, 薄新文. 基于cox2基因的细粒棘球绦虫环介导等温扩增检测方法的初步建立[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(2): 169-172.

Yan-yan ZHANG, Qian YE, Zheng-rong WANG, Xin-wen BO. Preliminary exploration of loop-mediated isothermal amplification based on cox2 gene of Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(2): 169-172.

图/表 2

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