中国人兽共患病学报2017，Vol.33Issue(5)：403-412,10.DOI:10.3969/j.issn.1002-2694.2017.05.004
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
Rapid detection of isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting curve analysis
摘要
Abstract
We detected the isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting (HRM) curve analysis and assessed the application value of the assay.The isoniazid resistance of 49 M.tuberculosis isolates preserved in laboratory was analyzed by the drug sensitivity test (traditional proportion method).Further analysis was made on the sequencing of the isoniazid resistance determining region in these test strains,and their mutation sites were screened.Specific primers used in the HRM curve analysis were designed based on the screened mutation sites,DNA mutations were assayed in the isoniazid-resistant gene determining region by the HRM curve analysis,and an assessment was made of the detection efficiency of the assay in isoniazid resistance in M.tuberculosis.Results of the drug sensitivity test (proportion method) showed that,of the 49 test strains,there were 20 isoniazid-resistant strains,29 isoniazid-sensitive strains.Results of the sequencing analysis showed that:1) KatG gene had four mutation patterns,i.e.,point mutations at site 234,at sites 234 and 315,at sites 234 and 463,and at sites 234,315 and 463;2) there were three mutations were detected in inhA gene,i.e.,mutations in inhA-8,-15 and-152.Analysis of gene mutation in drug-resistant strains found that of the 20 isoniazid-resistant strains,11 (55 ％) were mutated at codon 315 of KatG gene;6 (30％) were mutated in inhA-15 (4/20),-8 (1/20) and-153 (1/20) of inhA gene;two (10％) were mutated at codon 315 of KatG gene and in inhA-15;in one strain (5％),no mutation was detected in KatG and inhA genes.Through the gene mutation detection,the sensitivity and specificity of isoniazid resistance in M.tuberculosis were 95 ％ and 100 ％,respectively.Results of HRM curve analysis of drug-resistance gene mutations in test strains showed gene mutations were present in 18 strains and absent in 24 ones;referring to DNA sequencing results,the sensitivity and specificity of the assay were 94.7％ and 80％,respectively.Judged by mutations as drug-resistance via the HRM curve analysis,19 resistant and 24 sensitive strains were tested.With the drug sensitivity test results by the proportion method as controls,the sensitivity and specificity of the assay were 95 ％ and 82.76 ％,respectively.Use of the HRM curve in the detection of resistance of M.tuberculosis to isoniazid is characterized by good sensitivity and short time consuming,and has certain value in the rapid diagnosis of isoniazid-resistant tuberculosis.关键词
结核分枝杆菌/高分辨率熔解曲线(HRM)/DNA测序/耐药性/异烟肼Key words
Mycobacterium tuberculosis/high-resolution melting (HRM) curve/DNA sequencing/drug resistance/isoniazid分类
农业科技引用本文复制引用
杨彩虹,杨敏,于璐,包海洋,吴长新,曹旭东,陈创夫..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[J].中国人兽共患病学报,2017,33(5):403-412,10.基金项目
“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)Supported by the National Science and Technology Major Project of the 12th Five-Year Plan:"Prevention and Control of Severe Infectious Diseases Like AIDS and Viral Hepatitis" (No.2013ZX10003003-002) and the National Natural Science Foundation of China (No.31060333) （No.2013ZX10003003-002）
