Li Qiao, Wang Nenhan, Pang Mengdi, Miao Honghao, Dai Xiaowei, Li Bo, Yang Xinyu, Li Chuanyou, Liu Yi
Biobank of Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis & Thoracic Tumor Research Institute, Beijing 101149, China.
Beijing Center for Disease Prevention and Control, Beijing 100013, China.
Microorganisms. 2024 Jul 23;12(8):1507. doi: 10.3390/microorganisms12081507.
Tuberculosis (TB), a disease caused by (MTB) infection, remains a major threat to global public health. To facilitate early TB diagnosis, an IS6110 gene-based recombinase aided amplification (RAA) assay was coupled to a clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas13a fluorescence assay to create a rapid MTB detection assay (named RAA-CRISPR-MTB). Its diagnostic efficacy was evaluated for sensitivity and specificity through sequential testing of recombinant plasmids, mycobacterium strains, and clinical specimens. RAA-CRISPR detected IS6110 genes at levels approaching 1 copy/μL with pUC57-6110 as the template and 10 copies/μL with H37Rv as the template. There was no observed cross detection of non-tuberculosis mycobacteria (NTM) with either template. Furthermore, RAA-CRISPR testing of 151 clinical specimens yielded a diagnostic specificity rate of 100% and a diagnostic sensitivity rate of 69% that exceeded the corresponding Xpert MTB/RIF assay rate (60%). In conclusion, we established a novel RAA-CRISPR assay that achieved highly sensitive and specific MTB detection for use as a clinical TB diagnostic tool in resource-poor settings.
结核病（TB）是由结核分枝杆菌（MTB）感染引起的一种疾病，仍然是全球公共卫生的重大威胁。为促进结核病的早期诊断，基于IS6110基因的重组酶辅助扩增（RAA）检测与成簇规律间隔短回文重复序列（CRISPR）-Cas13a荧光检测相结合，创建了一种快速的MTB检测方法（命名为RAA-CRISPR-MTB）。通过对重组质粒、分枝杆菌菌株和临床标本进行连续检测，评估了其诊断效能的敏感性和特异性。以pUC57-6110为模板时，RAA-CRISPR检测IS6110基因的水平接近1拷贝/μL，以H37Rv为模板时为10拷贝/μL。两种模板均未观察到对非结核分枝杆菌（NTM）的交叉检测。此外，对151份临床标本进行RAA-CRISPR检测，诊断特异性率为100%，诊断敏感性率为69%，超过了相应的Xpert MTB/RIF检测率（60%）。总之，我们建立了一种新型的RAA-CRISPR检测方法，该方法实现了对MTB的高灵敏度和特异性检测，可作为资源匮乏地区临床结核病诊断工具。
