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Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis

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Abstract

Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis (TB), is the leading cause of death due to a single infectious agent worldwide. Rapid and accurate diagnosis of MTB is critical for controlling TB especially in resource-limited countries, since any diagnosis delay increases the chances of transmission. Here, a real-time recombinase-aided amplification (RAA) assay targeting conserved positions in IS1081 gene of MTB, is successfully established to detect MTB. The intact workflow was completed within 30 min at 42 °C with no cross-reactivity observed for non-tuberculous mycobacteria and other clinical bacteria, and the detection limit for recombinant plasmid of MTB IS1081 was 163 copies/reaction at 95% probability, which was approximately 1.5-fold increase in analytical sensitivity for the detection of MTB, compared to conventional quantitative real-time PCR (qPCR; 244 copies/reaction). Furthermore, the result of clinical performance evaluation revealed an increased sensitivity of RAA assay relative to qPCR was majorly noted in the specimens with low bacteria loads. Our results demonstrate that the developed real-time RAA assay is a convenient, sensitive, and low-cost diagnostic tool for the rapid detection of MTB.

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Data availability

The dataset analyzed in this study is available from the corresponding author on reasonable request.

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Funding

This study was supported by the Beijing Hospitals Authority Ascent Plan (DFL20191601), the Capital”s Funds for Health Improvement and Research (2020-1-1041), and the Beijing Hospitals Authority Clinical Medicine Development of Special Funding (ZYLX202122). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. Yuanyuan Liu·and Weicong Ren have contributed equally to this work.

Authors and Affiliations

  1. Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China

    Yuanyuan Liu, Weicong Ren, Zhongtan Xue, Wei Wang, Xuxia Zhang, Cong Yao, Yuanyuan Shang, Shanshan Li & Yu Pang

  2. Department of Research, Beijing Chest Hospital, Capital Medical University/Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing, China

    Yuedong Miao & Fengling Mi

Authors
  1. Yuanyuan Liu
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Contributions

Conceptualization, F. L .M. and Y. P.; methodology, Y. Y. L. and W. C. R.; software, Z. T. X.; validation, Y. D. M., W. W., and X. X .Z.; formal analysis, C. Y.; investigation, Y. Y. S.; resources, S. S. L.; data curation, Y. Y. L. and Y. P.; writing—original draft preparation, Y. Y. L. and Y. P.; writing—review and editing, Y. P.; visualization, W. C. R; supervision, F. L. M. and Y. P.; project administration, Y. P.; funding acquisition, Y. P. All the authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Fengling Mi or Yu Pang.

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Ethical approval

This study was conducted in accordance with the tenets of the World Medical Association’s Declaration of Helsinki and approved by the Ethics Committee of Beijing Chest Hospital, Capital Medical University (Approval No.: 2016KY005). Because this study only included demographic and clinical data anonymously, individual written consent was waived.

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Liu, Y., Ren, W., Xue, Z. et al. Real-time recombinase-aided amplification assay for rapid amplification of the IS1081 gene of Mycobacterium tuberculosis. Eur J Clin Microbiol Infect Dis 42, 963–972 (2023). https://doi.org/10.1007/s10096-023-04626-5

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