Experimental Research Center, Capital Institute of Pediatrics, Beijing, China.
Department of Clinical Laboratory, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, China.
Front Cell Infect Microbiol. 2023 May 23;13:1192134. doi: 10.3389/fcimb.2023.1192134. eCollection 2023.
Tuberculosis, caused by (MTB), is the second leading cause of death after COVID-19 pandemic. Here, we coupled multiple cross displacement amplification (MCDA) technique with CRISPR-Cas12a-based biosensing system to design a novel detection platform for tuberculosis diagnosis, termed MTB-MCDA-CRISPR. MTB-MCDA-CRISPR pre-amplified the specific gene of MTB by MCDA, and the MCDA results were then decoded by CRISPR-Cas12a-based detection, resulting in simple visual fluorescent signal readouts. A set of standard MCDA primers, an engineered CP1 primer, a quenched fluorescent ssDNA reporter, and a gRNA were designed targeting the gene of MTB. The optimal temperature for MCDA pre-amplification is 67°C. The whole experiment process can be completed within one hour, including sputum rapid genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), and CRISPR-Cas12a-gRNA biosensing process (5 minutes). The limit of detection (LoD) of the MTB-MCDA-CRISPR assay is 40 fg per reaction. The MTB-MCDA-CRISPR assay does not cross reaction with non-tuberculosis (NTM) strains and other species, validating its specificity. The clinical performance of MTB-MCDA-CRISPR assay was higher than that of the sputum smear microscopy test and comparable to that of Xpert method. In summary, the MTB-MCDA-CRISPR assay is a promising and effective tool for tuberculosis infection diagnosis, surveillance and prevention, especially for point-of-care (POC) test and field deployment in source-limited regions.
结核病由结核分枝杆菌（MTB）引起，是继 COVID-19 大流行之后的第二大致死病因。在这里，我们将多重交叉置换扩增（MCDA）技术与基于 CRISPR-Cas12a 的生物传感系统相结合，设计了一种用于结核病诊断的新型检测平台，称为 MTB-MCDA-CRISPR。MTB-MCDA-CRISPR 通过 MCDA 预扩增 MTB 的特异性基因，然后通过基于 CRISPR-Cas12a 的检测对 MCDA 结果进行解码，从而产生简单的可视荧光信号读数。一组标准的 MCDA 引物、一个工程化的 CP1 引物、一个淬灭荧光 ssDNA 报告分子和一个 gRNA 被设计用于靶向 MTB 的基因。MCDA 预扩增的最佳温度为 67°C。整个实验过程可在一个小时内完成，包括痰液快速基因组 DNA 提取（15 分钟）、MCDA 反应（40 分钟）和 CRISPR-Cas12a-gRNA 生物传感过程（5 分钟）。MTB-MCDA-CRISPR 检测的检测限（LoD）为每个反应 40 fg。MTB-MCDA-CRISPR 检测不会与非结核分枝杆菌（NTM）菌株和其他物种发生交叉反应，验证了其特异性。MTB-MCDA-CRISPR 检测的临床性能优于痰涂片显微镜检查，与 Xpert 方法相当。总之，MTB-MCDA-CRISPR 检测是一种有前途且有效的结核病感染诊断、监测和预防工具，特别是在资源有限地区的即时检测（POC）和现场部署。
