一株牛分枝杆菌的分离与鉴定

Isolation and Identification of a Mycobacterium Tuberculosis

采集一头检疫阳性的结核牛病变的淋巴组织进行细菌分离。分离物(Z-N)染色为阳性。提取细菌DNA,用PCR检测结核分枝杆菌特异性插入片段IS1081,确定该细菌为结核分枝杆菌。将PCR扩增出的特异性片段克隆到PMD-18T载体上。重组质粒经酶切和PCR鉴定均为阳性,测序结果与牛分枝杆菌标准菌株的同源性为100%。对其用Richard C.Huard等公布的PCR分型方法设计引物进行基因分型,确定该菌株为牛分枝杆菌BCG(M.bovis BCG)。

Through tuberculin skin test,comparative skin test and IFN-γ test,a positive Mycobacterium tuberculosis cattle was identified in the around of Urumqi region of Xinjang.Then bacterium was isolated from lymph nodes tissues with histological lesions respectively.One strain were isolated by culturing for 5 days and it is positive in Ziehl Neelsen.DNA of this strain was distilled by using DNeasy Tissue Handbook kit.According to the representative IS1081 insert fragment of Mycobacterium tuberculosis,a pair of primers was designed.And a Mycobacterium tuberculosis stain was identified by the profile of PCR products.Amplification product was cloned into the vetor PMD-18T.The results of recombinant plasmid enzyme digestion identification and PCR identification were the same as expected.The homology of the sequencing result and Mycobacterium bovis criteria strain was up to 100%.At last,according to Richard C.Huard,DNA of M.tuberculosis were used as templates and typing PCR test.The experimental results showed that it was M.bovis BCG.