Chang Yingchao, Zhang Mi, Liu Gaowen, Wu Xinlin, Yan Qiaolu, Yang Cuixian, Liu Li, Feng Yue, Xia Xueshan
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China.
Medical Laboratory, Yunnan Provincial Hospital of Infectious Disease, Kunming, Yunnan, China.
Microbiol Spectr. 2024 Oct 28;12(12):e0088724. doi: 10.1128/spectrum.00887-24.
Tuberculosis (TB) remains a major global health problem, and there is an urgent need for rapid, sensitive, and easy-to-use diagnostic technologies to improve TB diagnosis. In this study, we developed the nested multi-enzyme isothermal rapid amplification (nestMIRA) assay for TB. We designed several pairs of primers and probes targeting the IS6110 sequence of (Mtb) and performed combinatorial testing to optimize the performance of the TB nestMIRA assay. The reaction can be performed at a constant temperature of approximately 40°C and completed within 30 minutes in the same tube without opening the central cap. There has been no cross-reactivity with common (NTB) and respiratory pathogens. The TB nestMIRA assay has a minimum detection limit of 5 copies/μL for H37Rv genomic DNA and a limit of quantification of 100 CFU/ml. To test the diagnostic performance of the TB nestMIRA assay, we conducted a 163-person clinical cohort study using comprehensive reference standards as the gold standard for clinical diagnosis. Our study showed that TB nestMIRA performed slightly better than GeneXpert MTB/RIF (Xpert) (85.27% vs. 82.17%) and significantly better than culture (55.81%) and acid-fast bacillus (AFB) smear (38.76%). The TB nestMIRA assay offers speed, specificity, sensitivity, and convenience. We believe that it has the potential to be a rapid alternative for TB diagnosis, particularly in resource-limited settings.
In this study, we have successfully developed a method called nested multi-enzyme isothermal rapid amplification (nestMIRA) for the detection of (Mtb). This method involves a two-step thermostatic amplification process in the same tube and can be read using fluorescence and lateral flow dipstick (LFD) assays. It is known to be rapid, specific, and highly sensitive. Our method has shown promising results in the detection of clinical specimens, and we believe that it can be a valuable tool for the rapid detection of Mtb in a clinical setting.
结核病（TB）仍然是一个重大的全球健康问题，迫切需要快速、灵敏且易于使用的诊断技术来改善结核病诊断。在本研究中，我们开发了用于结核病的巢式多酶等温快速扩增（nestMIRA）检测方法。我们设计了几对靶向结核分枝杆菌（Mtb）IS6110序列的引物和探针，并进行组合测试以优化结核病nestMIRA检测方法的性能。该反应可在约40°C的恒定温度下进行，且在同一管中30分钟内完成，无需打开中心盖。与常见的非结核分枝杆菌（NTB）和呼吸道病原体无交叉反应。结核病nestMIRA检测方法对H37Rv基因组DNA的最低检测限为5拷贝/μL，定量限为100 CFU/ml。为了测试结核病nestMIRA检测方法的诊断性能，我们使用综合参考标准作为临床诊断的金标准进行了一项163人的临床队列研究。我们的研究表明，结核病nestMIRA的表现略优于GeneXpert MTB/RIF（Xpert）（85.27%对82.17%），且显著优于培养法（55.81%）和抗酸杆菌（AFB）涂片法（38.76%）。结核病nestMIRA检测方法具有速度快、特异性强、灵敏度高和便利性等优点。我们认为它有潜力成为结核病诊断的快速替代方法，特别是在资源有限的环境中。
在本研究中，我们成功开发了一种名为巢式多酶等温快速扩增（nestMIRA）的方法用于检测结核分枝杆菌（Mtb）。该方法在同一管中涉及两步恒温扩增过程，并且可以使用荧光和侧流试纸条（LFD）检测进行读取。已知其快速、特异且高度灵敏。我们的方法在临床标本检测中显示出有前景的结果，并且我们认为它可以成为临床环境中快速检测Mtb的有价值工具。
