Ren Weicong, Zhou You, Li Haoran, Shang Yuanyuan, Zhang Xuxia, Yuan Jinfeng, Li Shanshan, Li Chuanyou, Pang Yu
Department of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, China.
Chest Hospital of Guangxi Zhuang Autonomous Region, Liuzhou, Guangxi, China.
Front Microbiol. 2023 Feb 3;14:1117085. doi: 10.3389/fmicb.2023.1117085. eCollection 2023.
Tuberculosis diagnosis requires rapid, simple and highly sensitive methods. Clustered regularly interspaced short palindromic repeats (CRISPRs) and associated protein (Cas) systems are increasingly being used for clinical diagnostic applications, due to their high flexibility, sensitivity and specificity. We developed a sensitive (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens.
The conserved MTB IS1081 sequence was used to design CRISPR-derived RNAs (crRNAs) and T7 promoter sequencing-containing PCR primers for use in the CRISPR-MTB assay, then assay performance was evaluated using 401 clinical specimens.
The CRISPR-MTB assay provided a low limit of detection of 1 target sequence copy/μL and excellent specificity. Furthermore, use of the assay to detect MTB in bronchoalveolar lavage fluid (BALF), sputum and pus samples provided superior sensitivity (261/268, 97.4%) as compared to sensitivities of acid-fast bacilli (130/268, 48.5%) and mycobacterial culture (192/268, 71.6%) assays, and comparable or greater sensitivity to that of GeneXpert MTB/RIF (260/268, 97.0%).
The CRISPR-MTB assay, which provides excellent sensitivity and specificity for MTB detection in sputum, BALF and pus samples, is a viable alternative to conventional tests used to diagnose TB in resource-limited settings.
结核病诊断需要快速、简便且高度灵敏的方法。成簇规律间隔短回文重复序列（CRISPRs）及相关蛋白（Cas）系统因其高度的灵活性、敏感性和特异性，越来越多地被用于临床诊断应用。我们开发了一种灵敏的结核分枝杆菌（MTB）复合聚合酶链反应（PCR）-CRISPR/Cas13a检测方法（CRISPR-MTB），并评估了其在检测临床标本中MTB的性能。
使用保守的MTB IS1081序列设计CRISPR衍生RNA（crRNAs）和用于CRISPR-MTB检测的含T7启动子测序的PCR引物，然后使用401份临床标本评估检测性能。
CRISPR-MTB检测的检测下限低至1个靶序列拷贝/μL，特异性良好。此外，与抗酸杆菌检测（130/268，48.5%）和分枝杆菌培养检测（192/268，71.6%）相比，该检测方法用于检测支气管肺泡灌洗液（BALF）、痰液和脓液样本中的MTB时，灵敏度更高（261/268，97.4%），并且与GeneXpert MTB/RIF检测（260/268，97.0%）的灵敏度相当或更高。
CRISPR-MTB检测方法在痰液、BALF和脓液样本中检测MTB时具有出色的灵敏度和特异性，是资源有限环境中用于诊断结核病的传统检测方法的可行替代方案。
