焦磷酸测序技术快速检测结核分枝杆菌利福平耐药性研究

目的利用焦磷酸测序技术，建立一种高通量结核分枝杆菌利福平耐药性快速基因检测方法。方法应用生物素标记的引物扩增rpoB基因片段，经链亲和素标记的磁珠分离制备单链模板，设计2个测序引物在焦磷酸测序仪上检测rpoB基因耐药突变区的81bp序列突变情况，与H。Rv序列比对后判断耐药结果。结果通过建立的基于焦磷酸测序技术的结核分枝杆菌rpoB基因突变检测平台，32株利福平耐药株均在rpoB基因片段上检测到突变，22株利福平敏感株未检测到突变，检测结果与直接测序符合率达100％。结论本方法可快速、准确、高通量检测结核分枝杆菌耐利福平rpoB基因突变。

Detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing

Objective To develop an assay for rapid and sensitive detection of rifampin resistance in Mycobacterium tuberculosis by Pyrosequencing technology. Methods The rpoB gene was amplified by biotinylated primer. The PCR products were subjected to Pyrosequencing analysis using two different sequencing primers. All sequences obtained were gathered and compared in alignment with the rpoB sequence of H37Rv strain to detect the mutation. Results No mutation was found in 22 rifampin-sensitive strains ,while 32 rifampin-resistance strains had mutations in rifampin resistant determination region of rpoB gene. The results were confirmed by conventional DNA sequencing and the accordance rate was 100%. Condusion Pyrosequencing techology is a high throughput and high performance method in detecting mutations rifampin resistance in Mycobacterium tuberculosis.