利用插入序列IS6110建立结核分枝杆菌检测方法的研究

Research on establishing the detection method of Mycobacterium tuberculosis by inserting sequence IS6110

目的利用DNA聚合酶链式反应(PCR)扩增技术,建立一种敏感、特异的结核分枝杆菌检测方法,并探讨该方法在早期检测结核分枝杆菌中的应用价值。方法以结核分枝杆菌特异性较好的插入序列IS6110基因片段为目的序列,设计一对特异性引物,对结核分枝杆菌H37Rv、大肠杆菌、肠炎沙门菌、志贺菌、金黄色葡萄球菌、单增李斯特菌进行聚合酶链式反应扩增检测。结果只有结核分枝杆菌H37Rv菌株经聚合酶链反应扩增后可见247 bp的特异性条带,其余5种非结核分枝杆菌菌株扩增产物经电泳后均未出现特异性条带。敏感性实验可检测出10 fg结核分枝杆菌DNA。结论插入序列IS6110 DNA聚合酶链反应(PCR)是一种敏感、特异的结核分枝杆菌H37Rv的检测方法,为结核分枝杆菌的早期实验室检测提供了新的参考。

Objective To establish a sensitive, specific method for detection of Mycobacterium tuberculosis H37Rv by useing PCR, and discuss the application value in the early detection of Mycobacterium tuberculosis. Methods Design a pair of specific primers base on the inserted sequence IS6110 with high specificity of Mycobacterium tuberculosis. PCR amplification assays were performed for Mycobacterium tuberculosis H37Rv, E. coli, Salmonella enteritidis, Shigella, S. aureus and Listeria monocytogenes. Results 247 bp fragments were amplified only from Mycobacterium tuberculosis H37Rv and was not found in all nonmycobateria. Sensitivity test can detect 10 fg mycobacterium tuberculosis DNA. Conclusion Inserting sequence IS6110 PCR application is a sensitive, specific method, which provides a new reference for Mycobacterium tuberculosis detection in early laboratory tests.