荧光定量PCR探针熔解曲线法在结核分枝杆菌耐药基因检测中的应用

Fluorescence quantitative PCR probe melting curve method in detection of drug resistance genes in Mycobacterium tuberculosis

目的应用荧光定量PCR探针熔解曲线法检测耐多药结核病患者的临床分离菌株对一线抗结核药物的耐药基因突变。方法于2009年1月1日至2012年12月31日留取南京市胸科医院结核科门诊部或住院的所有肺结核患者痰液标本，每人3～5ml，均进行痰结核分枝杆菌培养、鉴定和药物敏感性试验（简称“药敏试验”），分离出结核分枝杆菌菌株67株。应用荧光定量PCR探针熔解曲线法，根据靶序列熔点变化，实时PCR检测结核病患者的临床分离标本对利福平、异烟肼、乙胺丁醇及链霉素的耐药情况；通过与传统药敏试验进行对比，对2种结果不一致的菌株进行基因测序分析，探讨其发生突变的位点差异，并观察在治疗中的变化。结果利福平和异烟肼的表型和基因型检测具有较高的一致性，分别为99％（66／67）和97％（65／67），不一致的菌株都是表型检测法为耐药，而基因型检测为敏感，测序未检测到突变。乙胺丁醇和链霉素的表型和基因型检测一致性较低，分别为60％（40／67）和75％（50／67）。测序发现，乙胺丁醇的突变发生在embB306和embB406位点上；链霉素突变发生在rrs基因517位点和907位点，以及rpsL43耐药密码子。结论应用荧光定量PCR探针熔解曲线法，能快速筛查结核分枝杆菌对一线抗结核药物的耐药情况。

Objective By fluorescence quantitative PCR probe melting curve method, to detect the resistance mutations of multidrug-resistant tuberculosis clinical isolates to first-line anti-tuberculosis drug, and to provide re- ference for multidrug-resistant tuberculosis treatment. Methods Collecting sputum of all TB patients visiting Nanjing chest hospital between January 1, 2009 and December 31, 2012, 3-- 5 ml per person, did culture and identi- fication of Mycobacterium tuberculosis and drug sensitivity test. Sixty seven strains of Mycobacterium tuberculosis were isolated. According to the target sequence changes in melting point, to test Rifampicin, Isoniazid, Ethambutol, and Streptomycin resistance by fluorescence quantitative PCR probe melting curve method. By comparison with the conventional susceptibility testing, to analyze the inconsistency of strain genome sequencing of 2 kinds of methods and explore the site differences in mutation, and observe the changes in treatment. Results The phenotypic and genotypic characteristics of the strains resisting to Rifampicin and lsoniazid had a high consistency, which was 99 （66/67） and 97% （65/67）, respectively. Testing different strains were phenotypic resistance but genotypic sensi- tive, which was not detected by sequencing mutation. However, the consistency between the phenotype and geno- type of anti-ethambutol and streptomycin strains was 600/00 （40/67） and 75% （50/67）, respectively. Sequencing found Ethambutol mutation occurred in the embB306 and embB406 sites. Streptomycin gene mutations occurred in rrs 517, rrs 907 and rpsL43. Conclusion The application of the fluorescence quantitative PCR probe melting curve can rapidly screen Mycobacterium tuberculosis drug resistance to first-line anti-tuberculosis drugs.