等位基因特异PCR技术检测结核分支杆菌rpoB基因突变研究

Development of an Allele Specific PCR for Detection of rpoB Gene Mutations Associated with the resistance of Myco-bactenum tuberculosis to Rifampin.

目的 建立并评价等位特异多聚酶链反应检测结核分支杆菌耐利福平基因rpoB突变的方法。方法 设计与rpoB基因突变后的碱基配对的引物用于扩增突变基因,建立AS-PCR检测rpoB基因突变的方法,并对58株结核菌进行了检测。结果AS-PCR检测结核菌耐利福平相关基因rpoB的敏感性为77.3％,特异性达91.7％。AS-PCR检测rpoB基因,有1628A→T、1657C→T,G和1673C→T突变分别占耐利福平结核菌株的18.2％,22.7％和36.4％。检测rpoB突变与MIC测定RFP耐药率和PCR-SSCP检测的总符合率分别为86.2％和74.1％。检测试验可在3～4h内完成。结论 AS-PCR方法是检测结核菌耐利福平基因突变的敏感和快速方法,可在临床实验室使用并为临床医师提供治疗依据。

Objective To establish and evaluate an allele - specific PCR method for rapid detection of rpoB gene mutations associated with M tuberculosis' s resistance to rifampin. Methods Based on the mutation mechanisms of rpoB, nucleotides containing mutated bases were employed in allele specific PCR. The results of AS- PCR detection were compared with these determined by DNA sequencing of rpoB and PCR - SSCP. Results The sensitivity and specificity of the AS - PCR we developed were 77.3% and 91. 7% respectively.The coincidence rates of AS - PCR and MIC and PCR- SSCP were 86.2% and 74.1% respectively.No significance was found when the results were compared between each two techniques. 17 out of 22 M tuberculosis strains resistant to RFP have been found mutations of 1628A→T, 1657C→T,G and 1673C→T by AS - PCR and rpoB sequencing. Conclusion The AS-PCR we established can be used in clinical laboratories for rapid detection of rpoB gene mutation, which can help doctors decide whether to put RFP into the treatment regimens.