Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex

Jing Xiao; Jieqiong Li; Shuting Quan; Yacui Wang; Guanglu Jiang; Yi Wang; Hairong Huang; Weiwei Jiao; Adong Shen

doi:10.3389/fbioe.2023.1233353

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex

Front Bioeng Biotechnol. 2023 Aug 25:11:1233353. doi: 10.3389/fbioe.2023.1233353. eCollection 2023.

Authors

Jing Xiao¹, Jieqiong Li², Shuting Quan¹, Yacui Wang¹, Guanglu Jiang³, Yi Wang⁴, Hairong Huang³, Weiwei Jiao¹, Adong Shen¹

Affiliations

¹ Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Key Laboratory of Major Diseases in Children, National Center for Children's Health, National Clinical Research Center for Respiratory Diseases, National Key Discipline of Pediatrics (Capital Medical University), Beijing Pediatric Research Institute, Ministry of Education, Beijing Children's Hospital, Capital Medical University, Beijing, China.
² Medical Research Center, Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, China.
³ National Tuberculosis Clinical Laboratory, Beijing Key Laboratory for Drug Resistance Tuberculosis Research, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing, China.
⁴ Experimental Research Center, Capital Institute of Pediatrics, Beijing, China.

Abstract

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies μL^-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

Keywords: CRISPR-Cas12a; Mycobacterium tuberculosis complex; fluorescent biosensor; lateral flow biosensor; multiplex detection; point-of-care testing.

Grants and funding

This study was supported by Beijing Municipal Natural Science Foundation (Grant number J200005), Capital Health Research and Development of Special Fund (Grant number 2020-2-1142), and Training Plan for High level Public Health Technical Talents (Grant number 2022-3-041).