建立和评估重叠延伸聚合酶链反应技术，用于快速有效地检测结核分枝杆菌的耐药性。

Establishment and evaluation of an overlap extension polymerase chain reaction technique for rapid and efficient detection of drug-resistance in Mycobacterium tuberculosis.

机构信息

Central Laboratory, Chongqing Public Health Medical Center, Chongqing, China.

Clinical Research Center, Chongqing Public Health Medical Center, Chongqing, China.

出版信息

Infect Dis Poverty. 2022 Mar 24;11(1):31. doi: 10.1186/s40249-022-00953-5.

DOI:10.1186/s40249-022-00953-5

PMID:35321759

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8942611/

Abstract

BACKGROUND

Rapid and accurate detection of drug resistance in Mycobacterium tuberculosis is critical for effective control of tuberculosis (TB). Herein, we established a novel, low cost strategy having high accuracy and speed for the detection of M. tuberculosis drug resistance, using gene splicing by overlap extension PCR (SOE PCR).

METHODS

The SOE PCR assay and Sanger sequencing are designed and constructed to detect mutations of rpoB, embB, katG, and inhA promoter, which have been considered as the major contributors to rifampicin (RFP), isoniazid (INH), and ethambutol (EMB) resistance in M. tuberculosis. One hundred and eight M. tuberculosis isolates came from mycobacterial cultures of TB cases at Chongqing Public Health Medical Center in China from December 2018 to April 2019, of which 56 isolates were tested with the GeneXpert MTB/RIF assay. Performance evaluation of the SOE PCR technique was compared with traditional mycobacterial culture and drug susceptibility testing (DST) or GeneXpert MTB/RIF among these isolates. Kappa identity test was used to analyze the consistency of the different diagnostic methods.

RESULTS

We found that the mutations of S531L, S315T and M306V were most prevalent for RFP, INH and EMB resistance, respectively, in the 108 M. tuberculosis isolates. Compared with phenotypic DST, the sensitivity and specificity of the SOE PCR assay for resistance detection were 100.00% and 88.00% for RFP, 94.64% and 94.23% for INH, and 68.97% and 79.75% for EMB, respectively. Compared with the GeneXpert MTB/RIF, the SOE PCR method was completely consistent with results of the GeneXpert MTB/RIF, with a concordance of 100% for resistance to RFP.

CONCLUSIONS

In present study, a novel SOE PCR diagnostic method was successfully developed for the accurate detection of M. tuberculosis drug resistance. Our results using this method have a high consistency with that of traditional phenotypic DST or GeneXpert MTB/RIF, and SOE PCR testing in clinical isolates can also be conducted rapidly and simultaneously for detection of drug resistance to RFP, EMB, and INH.

摘要

背景

快速准确地检测结核分枝杆菌的耐药性对于有效控制结核病（TB）至关重要。在此，我们建立了一种新颖的、低成本的策略，具有高准确性和速度，用于检测结核分枝杆菌的耐药性，使用基因拼接重叠延伸 PCR（SOE PCR）。

方法

设计和构建 SOE PCR 检测和 Sanger 测序，以检测 rpoB、embB、katG 和 inhA 启动子的突变，这些突变被认为是结核分枝杆菌利福平（RFP）、异烟肼（INH）和乙胺丁醇（EMB）耐药的主要贡献者。108 株结核分枝杆菌分离株来自中国重庆公共卫生医疗中心 2018 年 12 月至 2019 年 4 月的结核病例分枝杆菌培养物，其中 56 株分离株用 GeneXpert MTB/RIF 检测。对 SOE PCR 技术的性能评估与传统的分枝杆菌培养和药敏试验（DST）或 GeneXpert MTB/RIF 进行了比较。采用 Kappa 一致性检验分析不同诊断方法的一致性。

结果

我们发现，在 108 株结核分枝杆菌分离株中，S531L、S315T 和 M306V 突变最常见于 RFP、INH 和 EMB 耐药。与表型 DST 相比，SOE PCR 检测耐药性的敏感性和特异性分别为 100.00%和 88.00%（RFP）、94.64%和 94.23%（INH）、68.97%和 79.75%（EMB）。与 GeneXpert MTB/RIF 相比，SOE PCR 方法与 GeneXpert MTB/RIF 的结果完全一致，对 RFP 耐药的一致性为 100%。