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

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.

SUBMITTER: Xiao J

PROVIDER: S-EPMC10497956 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of <i>Mycobacterium tuberculosis</i> complex.

Xiao Jing J Li Jieqiong J Quan Shuting S Wang Yacui Y Jiang Guanglu G Wang Yi Y Huang Hairong H Jiao Weiwei W Shen Adong A

Frontiers in bioengineering and biotechnology 20230825

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 (IS<i>6110</i> an ...[more]

PMID: 37711452

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Project description:Tuberculosis (TB) is a chronic infectious disease with high mortality caused by the Mycobacterium tuberculosis complex (MTC). Its clinical symptoms include a prolonged cough with mucus, pleuritic chest pain, hemoptysis, etc., and predominant complications such as tuberculous meningitis and pleural effusion. Thus, developing rapid, ultrasensitive, and highly specific detection techniques plays an important role in controlling TB. Here, we devised CRISPR/CRISPR-associated 12b nuclease (CRISPR/Cas12b)-based multiple cross displacement amplification technique (CRISPR-MCDA) targeting the IS6110 sequence and used it to detect MTC pathogens. A newly engineered protospacer adjacent motif (PAM) site (TTTC) was modified in the linker region of the CP1 primer. In the CRISPR-MCDA system, the exponentially amplified MCDA amplicons with the PAM sites can guide the Cas12b/gRNA complex to quickly and accurately recognize its target regions, which successfully activates the CRISPR/Cas12b effector and enables ultrafast trans-cleavage of single-stranded DNA reporter molecules. The limit of detection of the CRISPR-MCDA assay was 5 fg/μL of genomic DNA extracted from the MTB reference strain H37Rv. The CRISPR-MCDA assay successfully detected all examined MTC strains and there was no cross-reaction with non-MTC pathogens, confirming that its specificity is 100%. The entire detection process can be completed within 70 min using real-time fluorescence analysis. Moreover, visualization detection (under UV light) was also designed to verify the results, eliminating the use of specialized instruments. In conclusion, the CRISPR-MCDA assay established in this report can be used as a valuable detection technique for MTC infection. IMPORTANCE The Mycobacterium tuberculosis complex pathogen is a crucial infectious agent of tuberculosis. Hence, improving the capability of MTC detection is one of the most urgently required strategies for preventing and controlling TB. In this report, we successfully developed and implemented CRISPR/Cas12b-based multiple cross displacement amplification targeting the IS6110 sequence to detect MTC pathogens. These results demonstrated that the CRISPR-MCDA assay developed in this study was a rapid, ultrasensitive, highly specific, and readily available method which can be used as a valuable diagnostic tool for MTC infection in clinical settings.

Dataset Information

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

Publications

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of <i>Mycobacterium tuberculosis</i> complex.

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