等位基因特异性PCR法快速检测结核分枝杆菌常见耐药突变基因  

Allele specific PCR for rapid detection of common gene mutations associated with the resistance of Mycobacterium tuberculosis

作　　者：彭婉婵[1] 刘文恩[1] 谭耀驹[2] 胡可[1] 蔡杏珊[2] 胡族琼[2] 李艳冰[1] 

机构地区：[1]中南大学湘雅医院检验科,长沙410008 [2]广州市胸科医院检验科,广州510095

出　　处：《临床检验杂志》2013年第4期249-252,共4页Chinese Journal of Clinical Laboratory Science

摘　　要：目的建立一套同步检测结核分枝杆菌(MTB)常见耐药突变基因的等位基因特异性PCR(AS-PCR)检测体系,以间接判断对利福平(RFP)与异烟肼(INH)的耐药性。方法用AS-PCR技术同步检测58株MTB临床分离株rpoB基因516位、526位和531位,katG基因315位及inhA基因-15位密码子突变,并与DNA测序结果进行比对。结果 AS-PCR法对RFP耐药株检出率为95.1%(39/41),其中rpoB基因531位、526位、516位点突变分别检出28株、10株、2株,包括531位与526位联合点突变1株;未检出突变的2株RFP耐药株经测序验证1株未发生突变、1株发生533位点突变;RFP敏感株均未检测到突变。AS-PCR法对INH耐药株检出率为86.5%(45/52),其中katG基因315位点突变43株、inhA基因-15位点突变2株,未检出突变的7株INH耐药株经测序验证未见突变;INH敏感株均未检测到突变。结论等位基因特异性PCR能够快速检测MTB常见突变基因,具有较高的敏感性与特异性,快速经济、操作简便。Objective To establish the allele specific PCR （AS-PCR） system for the detection of common gene mutations associated with tbe resistance of Mycobacterium tuberculosis （MTB）, and screen the resistance of MTB to rifampin （RFP） and isoniazid （INH）. Methods The mutations in codons 516, 526 and 531 of the rpoB gene, codon 351 of the katG gene, and the 15th nucleotide preced- ing the mabA-inhA operon from 58 MTB isolates were detected by the AS-PCR, and the obtained results were compared with that of DNA sequencing. Results The detection rate of RFP-resistant strains was 95.1% （39/41）, including 27 strains with rpoB-531 muta- tion, 9 with rpoB-526, 2 with rpoB-516, and 1 with double mutation in rpoB-531 and rpoB-526. The other 2 RFP-resistant strains, 1 without mutation and 1 with rpoB-533 mutation by sequencing, were not detected by the AS-PCR. The detection rate of INH-resistant strains was 86.5% （45/52）, including 43 with katG-315 mutation and 2 with inhA - 15. The other 7 INH-resistant strains undetected by the AS-PCR were demonstrated no mutation by sequencing. No mutation was detected in the strains sensitive to RFP or INH. Con- clusion The AS-PCR assay was a simple and rapid method with high sensitivity and specificity, which could detect the common gene mutations associated with the resistance of MTB.

关 键 词：结核分枝杆菌 RPOB基因 KATG基因 inhA基因 等位基因特异性扩增 

分 类 号：R378.911[医药卫生—病原生物学] Q503[医药卫生—基础医学]