A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis
ObjectivesDrug resistance in tuberculosis seriously affects the eradication of tuberculosis, and isoniazid resistance is the second most commonly observed drug resistance in patients with tuberculosis. Timely and accurate detection of isoniazid resistance is critical to the treatment of tuberculosis...
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Frontiers Media S.A.
2025-03-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1548965/full |
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| author | Zhiqiang Han Zhiqiang Han Zhiqiang Han Xichao Ou Ruiqing Zhang Xiaona Lv Xiaona Lv Xiaona Lv Yuxin Wang Yuxin Wang Yuxin Wang Hongyi Li Hongyi Li Hongyi Li Xinxin Shen Xuejun Ma Yanqing Tie |
| author_facet | Zhiqiang Han Zhiqiang Han Zhiqiang Han Xichao Ou Ruiqing Zhang Xiaona Lv Xiaona Lv Xiaona Lv Yuxin Wang Yuxin Wang Yuxin Wang Hongyi Li Hongyi Li Hongyi Li Xinxin Shen Xuejun Ma Yanqing Tie |
| author_sort | Zhiqiang Han |
| collection | DOAJ |
| description | ObjectivesDrug resistance in tuberculosis seriously affects the eradication of tuberculosis, and isoniazid resistance is the second most commonly observed drug resistance in patients with tuberculosis. Timely and accurate detection of isoniazid resistance is critical to the treatment of tuberculosis.MethodsA duplex one-step recombinase-aided PCR (DO-RAP) assay was developed for the rapid and sensitive detection of the katG Ser315Thr and inhA-15 (C-T) mutations in Mycobacterium tuberculosis, which are the most common isoniazid-resistant mutations. Quantitative recombinant plasmids were used to evaluate the sensitivity of DO-RAP, and 91 Mycobacterium tuberculosis strains with different genotypes, as well as 5 common respiratory tract bacteria, were used to evaluate the specificity of DO-RAP. A total of 78 sputum specimens were simultaneously detected using DO-RAP, quantitative PCR (qPCR) and sanger sequencing of nested PCR products. Sanger sequencing results were used as the standard to verify the clinical performance of DO-RAP.ResultsThe reaction time of DO-RAP was less than 1 h. The sensitivity of DO-RAP was 2 copies/reaction, which was 10 times higher than qPCR. The sensitivity of DO-RAP for detecting heterogenous resistance was 5%. There was no cross-reactivity between the isoniazid wild-type gene, drug-resistant mutant genes, and other common respiratory tract bacteria. Compared with Sanger sequencing, the sensitivity, specificity, PPV and NPV of DO-RAP were all 100%. There were 7 specimens with gray zone or negative qPCR results but positive DO-RAP test results.ConclusionThe DO-RAP can be adopted in ordinary qPCR equipment for the rapid, highly sensitive and specific detection of the isoniazid resistance genes of Mycobacterium tuberculosis. |
| format | Article |
| id | doaj-art-d508eba8059345e09b05c06d9342b3bd |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-d508eba8059345e09b05c06d9342b3bd2025-08-20T02:52:42ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-03-011610.3389/fmicb.2025.15489651548965A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosisZhiqiang Han0Zhiqiang Han1Zhiqiang Han2Xichao Ou3Ruiqing Zhang4Xiaona Lv5Xiaona Lv6Xiaona Lv7Yuxin Wang8Yuxin Wang9Yuxin Wang10Hongyi Li11Hongyi Li12Hongyi Li13Xinxin Shen14Xuejun Ma15Yanqing Tie16Hebei Medical University, Shijiazhuang, Hebei, ChinaDepartment of Clinical Laboratory, Hebei General Hospital, Shijiazhuang, Hebei, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaDepartment of Clinical Laboratory, Hebei General Hospital, Shijiazhuang, Hebei, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaHebei North University, Zhangjiakou, Hebei, ChinaHebei Medical University, Shijiazhuang, Hebei, ChinaDepartment of Clinical Laboratory, Hebei General Hospital, Shijiazhuang, Hebei, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaDepartment of Clinical Laboratory, Hebei General Hospital, Shijiazhuang, Hebei, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaHebei North University, Zhangjiakou, Hebei, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaNational Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, NHC Key Laboratory of Medical Virology and Viral Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, ChinaDepartment of Clinical Laboratory, Hebei General Hospital, Shijiazhuang, Hebei, ChinaObjectivesDrug resistance in tuberculosis seriously affects the eradication of tuberculosis, and isoniazid resistance is the second most commonly observed drug resistance in patients with tuberculosis. Timely and accurate detection of isoniazid resistance is critical to the treatment of tuberculosis.MethodsA duplex one-step recombinase-aided PCR (DO-RAP) assay was developed for the rapid and sensitive detection of the katG Ser315Thr and inhA-15 (C-T) mutations in Mycobacterium tuberculosis, which are the most common isoniazid-resistant mutations. Quantitative recombinant plasmids were used to evaluate the sensitivity of DO-RAP, and 91 Mycobacterium tuberculosis strains with different genotypes, as well as 5 common respiratory tract bacteria, were used to evaluate the specificity of DO-RAP. A total of 78 sputum specimens were simultaneously detected using DO-RAP, quantitative PCR (qPCR) and sanger sequencing of nested PCR products. Sanger sequencing results were used as the standard to verify the clinical performance of DO-RAP.ResultsThe reaction time of DO-RAP was less than 1 h. The sensitivity of DO-RAP was 2 copies/reaction, which was 10 times higher than qPCR. The sensitivity of DO-RAP for detecting heterogenous resistance was 5%. There was no cross-reactivity between the isoniazid wild-type gene, drug-resistant mutant genes, and other common respiratory tract bacteria. Compared with Sanger sequencing, the sensitivity, specificity, PPV and NPV of DO-RAP were all 100%. There were 7 specimens with gray zone or negative qPCR results but positive DO-RAP test results.ConclusionThe DO-RAP can be adopted in ordinary qPCR equipment for the rapid, highly sensitive and specific detection of the isoniazid resistance genes of Mycobacterium tuberculosis.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1548965/fullMycobacterium tuberculosisisoniazid resistance mutationrecombinant enzyme-assisted PCR (RAP)locked nucleic acid technology (LNA)highly sensitive |
| spellingShingle | Zhiqiang Han Zhiqiang Han Zhiqiang Han Xichao Ou Ruiqing Zhang Xiaona Lv Xiaona Lv Xiaona Lv Yuxin Wang Yuxin Wang Yuxin Wang Hongyi Li Hongyi Li Hongyi Li Xinxin Shen Xuejun Ma Yanqing Tie A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis Frontiers in Microbiology Mycobacterium tuberculosis isoniazid resistance mutation recombinant enzyme-assisted PCR (RAP) locked nucleic acid technology (LNA) highly sensitive |
| title | A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis |
| title_full | A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis |
| title_fullStr | A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis |
| title_full_unstemmed | A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis |
| title_short | A duplex one-step recombinase aided PCR assay for the rapid and sensitive detection of the isoniazid resistance genes katG and inhA in Mycobacterium tuberculosis |
| title_sort | duplex one step recombinase aided pcr assay for the rapid and sensitive detection of the isoniazid resistance genes katg and inha in mycobacterium tuberculosis |
| topic | Mycobacterium tuberculosis isoniazid resistance mutation recombinant enzyme-assisted PCR (RAP) locked nucleic acid technology (LNA) highly sensitive |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1548965/full |
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