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

发布时间：2018-02-15 08:23

本文关键词： 细粒棘球绦虫细胞色素c氧化酶亚基基因环介导等温扩增　出处：《中国寄生虫学与寄生虫病杂志》2017年02期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a safe, rapid and efficient method for the diagnosis of Echinococcus granulosus (Echinococcus granulosus) by loop-mediated isothermal amplification. Methods according to the sequence of cytochrome c oxidase subunit 2 cytochrome oxidase subunit 2cox2 of Echinococcus granulosus, Four LAMP primers were designed and LAMP detection method was established. Echinococcus granulosus was detected by LAMP method and routine PCR method. Nine species of DNA parasites, such as Cysticercus tenuicollisa, Moniezia expansa, Thysaniezia ovillailla, Avitellina centripunctata, flagella nematodesa, Fasciola sinensis, E. multilocularisia and Haemonchus contortus, et al. To verify the specificity of LAMP method; The sensitivity of standard plasmid of Echinococcus granulosus containing cox2 gene fragment in gradient dilution was detected by LAMP assay and conventional PCR method respectively. The positive rate of cox2 gene in Echinococcus granulosus was calculated by Lamp PCR and sandwich ELISA method. Results the specificity of LAMP method showed that, Only in the reaction system using Echinococcus granulosus DNA as template was the specific product. Lamp method could still show ladder bands when the standard plasmid concentration of Echinococcus granulosus was 16.09 g / 渭 l. The sensitivity of plasmid lamp assay with concentration above 16.9 fg / 渭 l was 1 000 times higher than that of conventional PCR method. The results of DNA detection for Echinococcus granulosus in 50 dog feces were the same as those detected by sandwich ELISA method. Conclusion the LAMP detection method based on the mitochondrial cox2 gene of Echinococcus granulosus is convenient, specific and sensitive. It is suitable for rapid detection of Echinococcus granulosus infected dogs.
【作者单位】：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;石河子大学;
【基金】：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)~~
【分类号】：R440;R532.32

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