Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex
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 ca...
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1233353/full |
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| author | Jing Xiao Jieqiong Li Shuting Quan Yacui Wang Guanglu Jiang Yi Wang Hairong Huang Weiwei Jiao Adong Shen |
| author_facet | Jing Xiao Jieqiong Li Shuting Quan Yacui Wang Guanglu Jiang Yi Wang Hairong Huang Weiwei Jiao Adong Shen |
| author_sort | Jing Xiao |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T12:16:25Z |
| format | Article |
| id | doaj.art-1df83e7943934efd8f626b8de0e894f2 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-12T12:16:25Z |
| publishDate | 2023-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-1df83e7943934efd8f626b8de0e894f22023-08-30T09:58:14ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-08-011110.3389/fbioe.2023.12333531233353Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complexJing Xiao0Jieqiong Li1Shuting Quan2Yacui Wang3Guanglu Jiang4Yi Wang5Hairong Huang6Weiwei Jiao7Adong Shen8Laboratory 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, ChinaMedical Research Center, Beijing Institute of Respiratory Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, ChinaLaboratory 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, ChinaLaboratory 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, ChinaNational 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, ChinaExperimental Research Center, Capital Institute of Pediatrics, Beijing, ChinaNational 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, ChinaLaboratory 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, ChinaLaboratory 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, ChinaSince 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.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1233353/fullMycobacterium tuberculosis complexCRISPR-Cas12amultiplex detectionfluorescent biosensorlateral flow biosensorpoint-of-care testing |
| spellingShingle | Jing Xiao Jieqiong Li Shuting Quan Yacui Wang Guanglu Jiang Yi Wang Hairong Huang Weiwei Jiao Adong Shen Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex Frontiers in Bioengineering and Biotechnology Mycobacterium tuberculosis complex CRISPR-Cas12a multiplex detection fluorescent biosensor lateral flow biosensor point-of-care testing |
| title | Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex |
| title_full | Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex |
| title_fullStr | Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex |
| title_full_unstemmed | Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex |
| title_short | Development and preliminary assessment of a CRISPR–Cas12a-based multiplex detection of Mycobacterium tuberculosis complex |
| title_sort | development and preliminary assessment of a crispr cas12a based multiplex detection of mycobacterium tuberculosis complex |
| topic | Mycobacterium tuberculosis complex CRISPR-Cas12a multiplex detection fluorescent biosensor lateral flow biosensor point-of-care testing |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1233353/full |
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