实时荧光定量聚合酶链反应结合高分辨率熔解曲线分析：一种用于筛查耐多药肺外结核的快速实用方法。

Real-time PCR followed by high-resolution melting curve analysis: A rapid and pragmatic approach for screening of multidrug-resistant extrapulmonary tuberculosis.

作者信息

Sharma Kusum, Sharma Megha, Singh Shreya, Modi Manish, Sharma Aman, Ray Pallab, Varma Subhash

机构信息

Department of Medical Microbiology, Department of Neurology and Department of Internal Medicine, Post Graduate Institute of Medical Education and Research, Chandigarh, India.

出版信息

Tuberculosis (Edinb). 2017 Sep;106:56-61. doi: 10.1016/j.tube.2017.07.002. Epub 2017 Jul 5.

DOI:10.1016/j.tube.2017.07.002

PMID:28802406

Abstract

INTRODUCTION

Multidrug resistance (MDR) in extrapulmonary tuberculosis (EPTB) is a diagnostic challenge in an endemic country like India. Timely detection of MDR-TB can contribute to a better patient outcome.

OBJECTIVE

To perform real-time PCR (qPCR) using rpoB, mpb64 and IS6110 gene on a variety of EPTB samples and to compare the performance of different gene targets. All qPCR positive samples were subjected to high resolution melt-curve analysis (HRM analysis) for rpoB and katG gene to evaluate its potential for MDR screening among different sample types.

METHODS

Real-time PCR using rpoB, mpb64 and IS6110 genes was carried out on 200 cases of study group and 100 cases of non-TB control group. The study group consisted of 100 culture-confirmed and 100 clinically suspected cases of EPTB. Phenotypic drug susceptibility testing (DST) for culture isolates was performed by the 1% indirect agar proportion method. DNA extracted from all qPCR positive samples was subjected to rpoB and katG HRM analysis for screening of MDR. Sequencing was used to confirm the results of HRM analysis and the results were also compared with phenotypic DST in all culture positive cases.

RESULTS

The sensitivity of qPCR using rpoB, mpb64 and IS6110 was 86.5%, 86.5% and 76.5%, respectively. All isolates from the control group were negative by all the three targets, giving a specificity of 100%. HRM analysis detected MDR in 22/200 (11%) isolates. 3/200 (1.5%) had mono-rifampicin resistance while 8/200 (4%) had mono-isoniazid resistance. HRM analysis identified an additional 4 MDR cases directly from the samples which were negative by culture. On sequencing, mutations were observed at codon 531 (60%); 533 (16%); 516 (12%) and 526 (12%) of the rpoB gene and at codon 315 (100%) of the katG gene. There was 100% concordance in the results of phenotypic DST, HRM analysis and sequencing.

CONCLUSION

The HRM analysis can play a promising role in the reliable and rapid screening of EPTB samples for detection of MDR.

摘要

引言

在印度这样的结核病流行国家，肺外结核病（EPTB）中的多药耐药（MDR）是一项诊断挑战。及时检测耐多药结核病有助于改善患者预后。

目的

对多种EPTB样本进行rpoB、mpb64和IS6110基因的实时荧光定量聚合酶链反应（qPCR），并比较不同基因靶点的性能。所有qPCR阳性样本均进行rpoB和katG基因的高分辨率熔解曲线分析（HRM分析），以评估其在不同样本类型中进行耐多药筛查的潜力。

方法

对200例研究组病例和100例非结核对照组病例进行rpoB、mpb64和IS6110基因的实时荧光定量聚合酶链反应。研究组包括100例培养确诊的EPTB病例和100例临床疑似病例。培养分离株的表型药物敏感性试验（DST）采用1%间接琼脂比例法进行。从所有qPCR阳性样本中提取的DNA进行rpoB和katG基因的HRM分析以筛查耐多药情况。测序用于确认HRM分析结果，并将所有培养阳性病例的结果与表型DST结果进行比较。

结果

使用rpoB、mpb64和IS6110基因的qPCR敏感性分别为86.5%、86.5%和76.5%。对照组的所有分离株在所有三个靶点检测中均为阴性，特异性为100%。HRM分析在22/200（11%）的分离株中检测到耐多药情况。3/200（1.5%）为单耐利福平，8/200（4%）为单耐异烟肼。HRM分析直接从培养阴性的样本中另外鉴定出4例耐多药病例。测序结果显示，rpoB基因的531密码子（60%）、533密码子（16%）、516密码子（12%）和526密码子（12%）以及katG基因的315密码子（100%）发生了突变。表型DST、HRM分析和测序结果的一致性为100%。