高分辨率熔解曲线技术用于结核分枝杆菌临床分离株异烟肼耐药性的快速检测

发布时间：2018-03-27 05:10

本文选题：结核分枝杆菌　切入点：高分辨率熔解曲线(HRM)　出处：《中国人兽共患病学报》2017年05期

【摘要】：目的评价高分辨率熔解曲线分析技术(high-resolution melting curve,HRM)检测结核分枝杆菌异烟肼耐药性的应用价值。方法 1)通过传统比例法药敏实验对本实验室保存的49株结核分枝杆菌进行异烟肼耐药性分析。2)进一步对该结核分枝杆菌进行异烟肼耐药相关基因KatG基因和inhA基因进行异烟肼耐药决定区测序分析,筛查突变位点。3)根据筛查到的突变位点设计高分辨率熔解曲线分析所用的特异性引物,对异烟肼耐药基因耐药决定区进行高分辨率熔解曲线分析检测DNA突变,评估用高分辨率熔解曲线分析技术对结核分枝杆菌异烟肼耐药性检测的效率。结果 1)比例法药敏结果显示,49株实验菌株中20株为异烟肼耐药株,29株为异烟肼敏感株。2)测序分析结果显示:(1)KatG基因出现了4种突变形式,分别是234单位点突变;234、315双位点突变;234、463双位点突变;234、315、463三位点联合突变。(2)inhA基因检测到3种突变,即-8位、-15位、-152位。3)(1)对耐药株的基因突变进行分析发现:20株异烟肼耐药株中11株存在KatG基因第315位密码子的突变、占异烟肼耐药株的55%;6株存在inhA基因-15(4株)位碱基、-8(1株)位碱基、-152(1株)位碱基的突变,共占异烟肼耐药株的30%;2株同时存在基因KatG315密码子和inhA-15位碱基的突变,占异烟肼耐药株的10%;1株均未检测到KatG基因和inhA基因的突变,占异烟肼耐药株的5%。通过基因突变检测结核分枝杆菌异烟肼耐药性的敏感性为95%、特异性为100%。(2)用高分辨率溶解曲线检测实验菌株耐药基因突变的结果显示,18株存在基因突变,24株不存在基因突变,检测的灵敏度为94.7%、特异性为80%。(3)以高分辨率熔解曲线检测到突变为耐药的判断标准,检出19株为耐药株,24株为敏感株。以比例法药敏结果为参照,检测的灵敏度为95%、特异性为82.76%。结论高分辨率溶解曲线用于结核分枝杆菌对异烟肼耐药性的检测具有较好的灵敏度,耗时短,在异烟肼耐药结核病的快速诊断方面具有一定的应用价值。
[Abstract]:Objective to evaluate the value of high-resolution melting curved-HRM in the detection of isoniazid resistance of Mycobacterium tuberculosis. Methods 1) 49 strains of Mycobacterium tuberculosis preserved in our laboratory were tested by traditional proportional susceptibility assay. Further analysis of isoniazid resistance-related gene KatG gene and inhA gene was carried out by isoniazid resistance analysis. 2) the sequence analysis of isoniazid resistance determining region was carried out for Mycobacterium tuberculosis. Screening mutation locus. 3) according to the specific primers designed for high resolution fusion curve analysis, the DNA mutation was detected by high resolution melting curve analysis of isoniazid resistance gene. To evaluate the efficiency of high resolution fusion curve analysis in the detection of isoniazid resistance of Mycobacterium tuberculosis. Results 1) the results of proportional drug sensitivity analysis showed that 20 of 49 experimental strains were isoniazid resistant and 29 were isoniazid. The sequence analysis of the susceptible strain. 2) showed that there were four mutations in the KatG gene. The 234315 double locus mutation and 234315463 triple locus combined mutation of 234315463 were identified as 234315463 and 234315463 / 234315463 / 234315463 / 234315463 / 234315463 / 234315463 respectively. The gene mutation of resistant strains was analyzed. It was found that 11 of 20 isoniazid resistant strains had codon 315 mutation of KatG gene. Among the isoniazid resistant strains, there were mutations of inhA codon and inhA-15 codon in 55 / 6 strains of isoniazid resistant strains (1 strain) and 1 strain (1 strain) at the base of 1 strain). In total, 30g / 2 strains of isoniazid resistance strains had mutations in the codon of KatG315 and the mutation of the inhA-15 site base at the same time, and there was no mutation in the nucleotide sequence of KatG315 codon and inhA-15 locus in the 6 strains of isoniazid resistance. No mutation of KatG gene or inhA gene was detected in 10 strains of isoniazid resistant strain. The sensitivity and specificity of detection of isoniazid resistance of Mycobacterium tuberculosis by gene mutation were 95 and 100% respectively. There was no gene mutation in 24 strains. The sensitivity of detection was 94.7 and the specificity was 80%.) according to the criteria of high resolution melting curve to detect mutation as drug resistance, 19 strains were identified as resistant strains and 24 strains as sensitive strains, and the results were compared with the results of proportional method. The sensitivity of detection is 95 and the specificity is 82.76.Conclusion the high resolution dissolution curve has good sensitivity and short time for the detection of isoniazid resistance of Mycobacterium tuberculosis. It has certain application value in fast diagnosis of isoniazid-resistant tuberculosis.
【作者单位】：石河子大学动物科技学院;石河子大学生命科学学院;石河子大学医学院;
【基金】：“十二五”国家科技重大专项(No.2013ZX10003003-002) 国家自然科学基金项目(No.31060333)~~
【分类号】：R440;R52

【相似文献】