评价针对三种不同结核分枝杆菌基因的环介导等温扩增检测方法的诊断价值。
Evaluation of the diagnostic value of loop-mediated isothermal amplification assays targeting three different Mycobacterium tuberculosis genes.
机构信息
Clinical Laboratory, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149, China.
Disease Prevention and Control Department, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing 101149,China.
出版信息
J Microbiol Methods. 2022 Sep;200:106547. doi: 10.1016/j.mimet.2022.106547. Epub 2022 Aug 1.
BACKGROUND
The aim of the study was to evaluate a loop-mediated isothermal amplification (LAMP) assay for the ability to diagnose tuberculosis directly from clinical samples rapidly.
METHODS
LAMP assays were performed using previously reported primer sets to amplify three specific Mycobacterium tuberculosis (MTB) gene targets, hspX, gyrB, and IS6110. Quantitated DNA from strain H37Rv were detected for assessment of analytical sensitivity; specificity was evaluated by testing eight species of non-tuberculosis Mycobacterium (NTM) and four unrelated bacterial species. Sputum samples from 68 pulmonary tuberculosis patients and a control group consisting of 45 lung cancer patients and 20 healthy controls were analyzed using LAMP assays, and then compared with smear, culture and quantitative real-time PCR (qRT-PCR) methods.
RESULTS
All three LAMP assays showed 100% specificity for MTB when tested against NTM and other bacterial species. The gyrB-LAMP assay was able to detect 60 cfu/ml of H37Rv suspension within 1 h, similar to qRT-PCR, but 10 times more sensitive than the hspX-LAMP and IS6110-LAMP assays. In clinical samples, when qRT-PCR was used as the reference method, the sensitivity of the three LAMP assays targeting hspX, gyrB, and IS6110 genes was 94.6, 98.2 and 92.9%, respectively.
CONCLUSIONS
LAMP is more sensitive than smear microscopy and close to qRT-PCR in sensitivity for the detection of MTB. LAMP has comparable specificity to qRT-PCR but was more rapid and convenient.
背景
本研究旨在评估环介导等温扩增（LAMP）检测方法能否快速直接从临床样本中诊断结核病。
方法
使用先前报道的引物组进行 LAMP 检测，以扩增三个特定的结核分枝杆菌（MTB）基因靶标，hspX、gyrB 和 IS6110。使用定量 H37Rv 菌株 DNA 进行分析灵敏度评估；通过检测八种非结核分枝杆菌（NTM）和四种非相关细菌物种评估特异性。使用 LAMP 检测方法分析了 68 例肺结核患者和对照组（45 例肺癌患者和 20 例健康对照者）的痰液样本，并与涂片、培养和定量实时 PCR（qRT-PCR）方法进行比较。
结果
三种 LAMP 检测方法在检测 NTM 和其他细菌物种时对 MTB 均具有 100%的特异性。gyrB-LAMP 检测法在 1 小时内能够检测到 60 cfu/ml 的 H37Rv 悬浮液，与 qRT-PCR 相似，但比 hspX-LAMP 和 IS6110-LAMP 检测法敏感 10 倍。在临床样本中，当 qRT-PCR 作为参考方法时，针对 hspX、gyrB 和 IS6110 基因的三种 LAMP 检测方法的灵敏度分别为 94.6%、98.2%和 92.9%。
结论
LAMP 比涂片显微镜更敏感，与 qRT-PCR 相比，LAMP 对 MTB 的检测灵敏度接近。LAMP 与 qRT-PCR 具有相当的特异性，但更快速、方便。
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