基于PARMS技术的结核分枝杆菌利福平耐药基因rpoB分子标记的开发  

Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology

作　　者：刘晓慧 祖向阳 王珍珍[2] 赵朋超[1] 高蕾 赵战勤[3] 薛云[1] Xiaohui Liu;Xiangyang Zu;Zhenzhen Wang;Pengchao Zhao;Lei Gao;Zhanqin Zhao;Yun Xue(College of Medical Technology and Engineering,Henan University of Science and Technology,Luoyang 471023,Henan,China;Department of Laboratory Medicine,the First Affiliated Hospital of Henan University of Science and Technology,Luoyang 471023,Henan,China;College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,Henan,China)

机构地区：[1]河南科技大学医学技术与工程学院,河南洛阳471023 [2]河南科技大学第一附属医院检验科,河南洛阳471023 [3]河南科技大学动物科技学院,河南洛阳471023

出　　处：《生物工程学报》2021年第7期2503-2512,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.31672530,32072899,U1704117);河南省科技攻关项目(No.192102310037);河南科技大学研究生创新基金(No.CXJJ-2019KJ04)资助。

摘　　要：文中旨在建立结核分枝杆菌利福平耐药基因rpoB的荧光分子标记,为分子药物敏感性试验提供简便、可靠的基因分型检测方法。对比分析利福平耐药菌株rpoB基因531、526、516、511、513等氨基酸位点的基因突变与敏感菌株中等位基因的序列差异,结合PARMS技术(Penta-primer amplification refractory mutation system),建立rpoB基因的荧光分子标记。利用其对104例结核分枝杆菌临床分离株进行检测,经Sanger测序验证,正确率100%。并采用比例法药敏试验对这104份样本进行了利福平耐药性鉴定,与分子标记结果相符率为94.23%。结果表明,分子标记有较强可靠性,能检出表型药敏无法测出的低浓度耐药样本(511/533单位点突变)。建立的11组荧光分子标记能覆盖92%–96%的利福平耐药菌株rpoB基因突变类型,为快速检测结核分枝杆菌利福平耐药提供新思路。The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique(Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples(511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%–96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 利福平耐药 RPOB基因 PARMS技术 比例法药敏 

分 类 号：R378.911[医药卫生—病原生物学]