牛副结核病PCR检测方法的建立

根据GenBank上公布的副结核分枝杆菌C-2染色体的ISMav2基因序列设计特异性引物,对牛副结核分枝杆菌进行PCR扩增并将产物克隆到pMD18-T载体后测序。结果表明,扩增的目的片段大小为246 bp,与预期扩增序列同源性为99.6%。该PCR检测体系的特异性强,不能在非副结核分枝杆菌 DNA中扩增出条带;敏感性高,最低检测的DNA含量为1 pg。该检测体系的成功构建为牛副结核病的检测、鉴定和流行病学调查提供了有力的技术支持。

Establishment of PCR for Detecting Bovine Paratuberculosis

A pair of primers were designed according to the sequence of specific Mycobacterium paratuberculosis gene of C-2 chromosome(ISMav2),the ISMav2 gene was amplified by PCR and cloned into pMD18-T vector and then the gene sequence was detected.The results showed that the target gene band at a length 246 bp was amplified by PCR,with a homology of 99.6% to the gene sequence reported in GenBank.The experiments had proved that PCR assay possessed a high specificity,DNA bands couldn’t be amplified in other bacterial except Mycobacterium paratuberculosis.And the sensitivity test results indicated that PCR assay was more sensitive,which could detect ISMav2 with only 1 pg DNA.The successfully construction of the PCR assay provides strongly technical support for detection,identification and epidemiological investigations of bovine paratuberculosis.