Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens
ObjectiveTuberculosis 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 a...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-02-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1117085/full |
| _version_ | 1811173458566971392 |
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| author | Weicong Ren You Zhou Haoran Li Yuanyuan Shang Xuxia Zhang Jinfeng Yuan Shanshan Li Chuanyou Li Chuanyou Li Yu Pang |
| author_facet | Weicong Ren You Zhou Haoran Li Yuanyuan Shang Xuxia Zhang Jinfeng Yuan Shanshan Li Chuanyou Li Chuanyou Li Yu Pang |
| author_sort | Weicong Ren |
| collection | DOAJ |
| description | ObjectiveTuberculosis 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 Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens.MethodsThe 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.ResultsThe 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%).ConclusionThe 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. |
| first_indexed | 2024-04-10T17:47:14Z |
| format | Article |
| id | doaj.art-1dfa441b8478466cb5481fe2f778319e |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-10T17:47:14Z |
| publishDate | 2023-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-1dfa441b8478466cb5481fe2f778319e2023-02-03T04:54:39ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2023-02-011410.3389/fmicb.2023.11170851117085Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimensWeicong Ren0You Zhou1Haoran Li2Yuanyuan Shang3Xuxia Zhang4Jinfeng Yuan5Shanshan Li6Chuanyou Li7Chuanyou Li8Yu Pang9Department of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaChest Hospital of Guangxi Zhuang Autonomous Region, Liuzhou, Guangxi, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaDepartment of Tuberculosis, Beijing Center for Disease Prevention and Control, Beijing, ChinaDepartment of Bacteriology and Immunology, Beijing Chest Hospital, Beijing Tuberculosis and Thoracic Tumor Research Institute, Capital Medical University, Beijing, ChinaObjectiveTuberculosis 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 Mycobacterium tuberculosis (MTB) complex polymerase chain reaction (PCR)-CRISPR/Cas13a detection method (CRISPR-MTB) and then evaluated its performance in detecting MTB in clinical specimens.MethodsThe 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.ResultsThe 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%).ConclusionThe 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.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1117085/fulltuberculosisMycobacterium tuberculosis complex (MTB)GeneXpert MTB/RIFCRISPRPCRdiagnosis |
| spellingShingle | Weicong Ren You Zhou Haoran Li Yuanyuan Shang Xuxia Zhang Jinfeng Yuan Shanshan Li Chuanyou Li Chuanyou Li Yu Pang Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens Frontiers in Microbiology tuberculosis Mycobacterium tuberculosis complex (MTB) GeneXpert MTB/RIF CRISPR PCR diagnosis |
| title | Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens |
| title_full | Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens |
| title_fullStr | Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens |
| title_full_unstemmed | Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens |
| title_short | Development and clinical evaluation of a CRISPR/Cas13a-based diagnostic test to detect Mycobacterium tuberculosis in clinical specimens |
| title_sort | development and clinical evaluation of a crispr cas13a based diagnostic test to detect mycobacterium tuberculosis in clinical specimens |
| topic | tuberculosis Mycobacterium tuberculosis complex (MTB) GeneXpert MTB/RIF CRISPR PCR diagnosis |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2023.1117085/full |
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