Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification
ABSTRACT Background Tuberculosis (TB) remains a leading cause of mortality worldwide, particularly in developing nations. Currently, available diagnostic methods are often too costly or insufficiently sensitive for effective use in low‐ and middle‐income countries. Developing a rapid, convenient, an...
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Wiley
2025-06-01
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| Online Access: | https://doi.org/10.1002/ila2.70015 |
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| author | Xiaoyan Tan Yongcong Li Xufu Xiang Wei Wu Gang Wang Sai Zhang |
| author_facet | Xiaoyan Tan Yongcong Li Xufu Xiang Wei Wu Gang Wang Sai Zhang |
| author_sort | Xiaoyan Tan |
| collection | DOAJ |
| description | ABSTRACT Background Tuberculosis (TB) remains a leading cause of mortality worldwide, particularly in developing nations. Currently, available diagnostic methods are often too costly or insufficiently sensitive for effective use in low‐ and middle‐income countries. Developing a rapid, convenient, and accurate method for detecting the Mycobacterium tuberculosis complex (MTBC) is crucial to curtail the spread of TB. Methods Primers and probes targeting conserved regions of IS1081 were designed, and the RNase P gene was introduced as an internal control to prevent false‐negative results. M. tuberculosis control was used to optimize the reaction temperature. Additionally, we calculated and compared the limit of detection, specificity, and coincidence rate between this platform and the TaqMan real‐time fluorescence quantification method (RT‐qPCR) using two sets of national reference panels and 10 strains of MTBC. Results An on‐site MTBC‐multiplex recombinase polymerase amplification (MTBC‐mRPA) platform was established, with detection within 30 min over a broad temperature range (25°C–45°C). Probit analysis estimated a 95% limit of detection of 557.16 (95% confidence interval: 406.76–1062.67) bacteria/mL (p < 0.0001), close to the limit of detection of 461.84 (95% confidence interval: 342.55–881.57) bacteria/mL (p < 0.0001) of qPCR. The platform differentiated between non‐tuberculous mycobacteria and other common respiratory bacteria, showing 100% specificity. The coincidence rate between multiplex real‐time recombinase polymerase amplification (RT‐mRPA) and RT‐qPCR was 100%, indicating substantial similarity. Conclusion A simple, rapid, and visual MTBC‐mRPA platform coupled with rapid DNA extraction was developed for sensitive and specific detection of MTBC, especially suitable for on‐site screening of TB in low‐resource settings. |
| format | Article |
| id | doaj-art-ddb419ce19f94f4aa7b70887a38fa9d6 |
| institution | Kabale University |
| issn | 2834-443X 2834-4448 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Wiley |
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| series | iLabmed |
| spelling | doaj-art-ddb419ce19f94f4aa7b70887a38fa9d62025-08-20T03:40:13ZengWileyiLabmed2834-443X2834-44482025-06-013217118110.1002/ila2.70015Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase AmplificationXiaoyan Tan0Yongcong Li1Xufu Xiang2Wei Wu3Gang Wang4Sai Zhang5College of Pharmacy Shenzhen Technology University Shenzhen ChinaSchool of Life Science and Technology Southeast University Nanjing ChinaThe Key Laboratory for Biomedical Photonics of MOE at Wuhan National Laboratory for Optoelectronics‐Hubei Bioinformatics and Molecular Imaging Key Laboratory, Systems Biology Theme Department of Biomedical Engineering College of Life Science and Technology Huazhong University of Science and Technology Wuhan ChinaDepartment of Radiation Oncology Stanford University Stanford California USAGuangdong Provincial Key Laboratory of Medical Immunology and Molecular Diagnostics Department of Clinical Immunology Institute of Laboratory Medicine School of Medical Technology Guangdong Medical University Dongguan ChinaSchool of Life Science and Technology Southeast University Nanjing ChinaABSTRACT Background Tuberculosis (TB) remains a leading cause of mortality worldwide, particularly in developing nations. Currently, available diagnostic methods are often too costly or insufficiently sensitive for effective use in low‐ and middle‐income countries. Developing a rapid, convenient, and accurate method for detecting the Mycobacterium tuberculosis complex (MTBC) is crucial to curtail the spread of TB. Methods Primers and probes targeting conserved regions of IS1081 were designed, and the RNase P gene was introduced as an internal control to prevent false‐negative results. M. tuberculosis control was used to optimize the reaction temperature. Additionally, we calculated and compared the limit of detection, specificity, and coincidence rate between this platform and the TaqMan real‐time fluorescence quantification method (RT‐qPCR) using two sets of national reference panels and 10 strains of MTBC. Results An on‐site MTBC‐multiplex recombinase polymerase amplification (MTBC‐mRPA) platform was established, with detection within 30 min over a broad temperature range (25°C–45°C). Probit analysis estimated a 95% limit of detection of 557.16 (95% confidence interval: 406.76–1062.67) bacteria/mL (p < 0.0001), close to the limit of detection of 461.84 (95% confidence interval: 342.55–881.57) bacteria/mL (p < 0.0001) of qPCR. The platform differentiated between non‐tuberculous mycobacteria and other common respiratory bacteria, showing 100% specificity. The coincidence rate between multiplex real‐time recombinase polymerase amplification (RT‐mRPA) and RT‐qPCR was 100%, indicating substantial similarity. Conclusion A simple, rapid, and visual MTBC‐mRPA platform coupled with rapid DNA extraction was developed for sensitive and specific detection of MTBC, especially suitable for on‐site screening of TB in low‐resource settings.https://doi.org/10.1002/ila2.70015IS1081 geneMycobacterium tuberculosis complexRT‐mRPAtuberculosis |
| spellingShingle | Xiaoyan Tan Yongcong Li Xufu Xiang Wei Wu Gang Wang Sai Zhang Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification iLabmed IS1081 gene Mycobacterium tuberculosis complex RT‐mRPA tuberculosis |
| title | Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification |
| title_full | Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification |
| title_fullStr | Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification |
| title_full_unstemmed | Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification |
| title_short | Rapid and Visual Detection of Mycobacterium tuberculosis Complex Based on Multiplex Recombinase Polymerase Amplification |
| title_sort | rapid and visual detection of mycobacterium tuberculosis complex based on multiplex recombinase polymerase amplification |
| topic | IS1081 gene Mycobacterium tuberculosis complex RT‐mRPA tuberculosis |
| url | https://doi.org/10.1002/ila2.70015 |
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