快速检测双芽巴贝斯虫LAMP方法的建立  

Rapid Detection of Babesia bigemina by Loop-mediated Isothermal Amplification

作　　者：王素华 蔡渭明 李群[3] 杜爱芳[3] 

机构地区：[1]温州出入境检验检疫局,浙江温州325027 [2]绍兴出入境检验检疫局,浙江绍兴312000 [3]浙江大学动物预防医学研究所,浙江杭州310029

出　　处：《中国畜牧兽医》2011年第11期113-116,共4页China Animal Husbandry & Veterinary Medicine

基　　金：国家质检总局立项项目(2009IK007)

摘　　要：为提高双芽巴贝斯虫(Babesia bigemina)检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的B.bigemina检测方法。根据GenBank上公布的Babesia bigemina细胞色素b(Cytochrome b,cyt b)基因序列,设计4条特异地识别B.bigemina的cyt b基因6个特殊区域的LAMP引物,优化反应体系和条件,在Bst DNA聚合酶的作用下,65℃反应60min,加入SYBR GreenⅠ后观察。结果表明,该LAMP检测方法特异性强,与牛巴贝斯虫(Babesia bovis)等DNA不发生交叉反应;敏感性高,对B.bigemina的cyt b基因最小检测值为0.085fg/μL,是一般PCR方法的1000倍。该方法具有简单、快速、低成本的特点,可用于B.bigemina的基层现场快速检测。A loop-mediated isothermal amplification（LAMP） assay for rapid detection of Babesia bigemina was developed using primers specific to six distinct regions of Cytochrome b gene of Babesia bigemina.LAMP was performed using Bst DNA polymerase at 65 ℃ in water bath for 60 min and amplification results was visualized with SYBR Green Ⅰ.Specific bands could be detected for Babesia bigemina,but not for others.The detection limit of the assay was 0.085 fg/μL for Babesia bigemina,which was up to 1000 times higher than that of PCR methods.The LAMP methods was rapid and could be easily performed at a low cost.

关 键 词：双芽巴贝斯虫 环介导等温扩增技术 检测 CYTB基因 

分 类 号：S855.9[农业科学—临床兽医学]