牛巴贝斯虫巢式PCR诊断方法的建立

Development of nested-PCR assay for detecting Babesia bovis

根据GenBank发表的XJ-MSA-2c核苷酸序列(登录号:EU328267)设计的2对特异性引物MS-1、MS-2、MS-3以及MS-4,建立牛巴贝斯虫病巢式PCR快速检测方法。在特异性检测试验中,仅从MSA-2c质粒样本中扩增出622、350bp2条目的片段,与预期片段大小相符,而作为对照样本的双芽巴贝斯虫、牛环形泰勒虫、东方巴贝斯虫基因组DNA均无此扩增目的条带出现。第1次和第2次扩增的敏感性分别为1.75、1.75×10-2μg/L。在对46份全血的DNA样本巢式PCR和显微镜检测中,阳性检出率分别为34.8%(16/46)和23.9%(11/46)。结果表明,所建立的巢式PCR方法准确、敏感、特异,作为牛巴贝斯虫病的快速检测和小范围的流行病学调查,具有重要的临床意义。

In order to develop the nested-PCR assay for rapidly detecting Babesia bovis,four specific primers,MS-1,MS-2,MS-3 and MS-4,were designed according to the published XJ-MSA-2c of Babesia bovis in GenBank（accession number EU328267）.By the specific assay of nested-PCR,622bp and 350bp DNA fragments,which agree with the expective length,were amplified from samples of MSA-2c plasmid of Babeisa bovis,and were not found from the samples of Babesia bigemina,Bovine theileria annulata and Babesia orientalis.Sensitivity of the first and second amplifications by nested-PCR was 1.75 μg/L and 1.75×10^-2μg/L,respectively.In the DNA test by the nested-PCR and the test of Babeisa bovis by microscope in 46 blood samples,the positive rates were 16/46（34.8%）and 11/46（23.9%） respectively.The results showed the established method of nested-PCR assay was a precise,sensitive and specific for the rapid detection of Babesia bovis and the small scale of epidemiological investigation.