快速检测牛巴贝斯焦虫LAMP方法的建立

Rapid detection of Babesia bovis by loop-mediated isothermal amplification

为提高牛巴贝斯焦虫(Babesia bovis)病检出率,本研究采用环介导等温扩增技术(LAMP)建立一种快速、灵敏、特异的检测B.bovis的方法。根据GenBank中登录的B.bovis细胞色素b基因(cyt b)序列,设计4条LAMP引物,优化反应体系条件,在Bst DNA聚合酶的作用下,62℃反应60min,加入EvaGreen后,肉眼观测。结果表明,该LAMP检测体系特异性强,与双芽巴贝斯焦虫(B.bigemina)DNA等不发生交叉反应;敏感性高,最小检测值为0.014fg(相当于1.58×10-3虫体的拷贝数),为一般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.bovis病的现场快速检测。

A loop-mediated isothermal amplification（LAMP） assay for rapid detection of Babesia bovis was developed using primers specific to six distinct regions of Cytochrome b gene of Babesia bovis.LAMP was performed using Bst DNA polymerase at 62 ℃ in a water bath for 60 min and amplification results was visualized with EvaGreenⅠ.Specific bands could be detected for Babesia bovis,but not Babesia bigemina.The detection limit of the assay was 0.014 fg/μL for Babesia bovis,which was up to 1,000 times higher than that of PCR-based methods.The LAMP method was rapid and could be easily performed at a low cost.