牛卵形巴贝斯虫PCR检测方法的建立及初步应用

Establishment of PCR assay for detection of Babesia ovata

为建立牛卵形巴贝斯虫(B.ovata)快速检测方法,本研究根据GenBank中登录的B.ovata CCTη基因序列设计引物,建立了PCR检测方法。对该方法的最佳反应条件进行优化,并进行特异性、敏感性及临床样本检测试验。结果表明,建立的PCR方法扩增B.ovata CCTη基因片段大小为1 008 bp,与参考株序列同源性为100%。该方法对牛巴贝斯虫、牛双芽巴贝斯虫、牛瑟氏泰勒虫基因组DNA扩增结果均为阴性。最低可以检测样品中34个拷贝的DNA。通过对49份临床样本的检测,该方法比姬姆萨染色镜检阳性率高8.2%。本实验为B.ovata的诊断提供了一种特异、敏感的检测技术。

To establish a rapid diagnosis method for Babesia ovata, the PCR assay was developed with the specific primers were designed according to the sequence of the CCTη gene available in the GenBank. Under the optimized reaction conditions, the PCR assay was highly specific to amplify a 1,008 bp fragment from B. ovate genomic DNA with a detection limit of 34 copies, but no amplification with the DNA of B. bovis, B. bigemina and Theileria sergenti. Furthermore, a total of 49 blood DNA samples were tested by the PCR method and the results confirmed that the PCR method was sensitive and specific for detecting B. ovata infection in cattle.