结核分枝杆菌
英哈
异烟肼
数字聚合酶链反应
抗药性
rpoB公司
桑格测序
痰
肺结核
生物
分子生物学
分析灵敏度
聚合酶链反应
微生物学
病毒学
突变
医学
病理
遗传学
基因
替代医学
作者
Siqi Zhang,Xiaohong Chen,Zhihong Lin,Yi Tan,Bin Liang,Yamin Pan,Minghui Huang,Biyi Su,Xiaoman Hu,Ye Xu,Qingge Li
摘要
The quantitative detection of drug-resistance mutations in Mycobacterium tuberculosis (MTB) is critical for determining the drug resistance status of a sample. We developed a drop-off droplet digital PCR (ddPCR) assay targeting all major isoniazid (INH)-resistant mutations. The ddPCR assay consisted of three reactions: reaction A detects mutations at katG S315; reaction B detects inhA promoter mutations; and reaction C detects ahpC promoter mutations. All reactions could quantify 1%-50% of mutants in the presence of the wild-type, ranging from 100 to 50,000 copies/reaction. Clinical evaluation with 338 clinical isolates yielded clinical sensitivity of 94.5% (95% confidence interval [CI] = 89.1%-97.3%) and clinical specificity of 97.6% (95% CI = 94.6%-99.0%) compared with the traditional drug susceptibility testing (DST). Further clinical evaluation using 194 nucleic acid-positive MTB sputum samples revealed clinical sensitivity of 87.8% (95% CI = 75.8%-94.3%) and clinical specificity of 96.5% (95% CI = 92.2%-98.5%) in comparison with DST. All the mutant and heteroresistant samples detected by the ddPCR assay but susceptible by DST were confirmed by combined molecular assays, including Sanger sequencing, mutant-enriched Sanger sequencing and a commercial melting curve analysis-based assay. Finally, the ddPCR assay was used to monitor longitudinally the INH-resistance status and the bacterial load in nine patients undergoing treatment. Overall, the developed ddPCR assay could be an indispensable tool for quantification of INH-resistant mutations in MTB and bacterial loads in patients.
科研通智能强力驱动
Strongly Powered by AbleSci AI