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<p>The global management of tuberculosis (TB) is limited by the infrastructure requirements of current molecular diagnostics and the logistical burdens of sputum-based testing. While isothermal nucleic acid amplification provides a decentralized alternative, its clinical utility is often limited by amplicon carryover contamination and complex multi-step protocols. In this study we developed a fully integrated, “one-pot” biplex assay that couples recombinase polymerase amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‑associated protein Cas12a-mediated <i>trans</i>-cleavage within a single, homogeneous reaction. This single-pot chemistry that targets the IS<i>1081</i> and IS<i>6110</i> genes of <i>Mycobacterium tuberculosis</i> eliminates the need for post-amplification handling, addressing a primary failure mode of decentralized testing, while maintaining an analytical sensitivity of 1.1 genomes per 15-µL reaction. The assay delivers sample-to-result within 30&#xa0;min, with no observed cross-reactivity with common nontuberculous mycobacteria and respiratory pathogens. We further validated the system using clinical tongue swab samples, a non-invasive sampling method recently recognized by the World Health Organization to improve diagnostic yield in sputum-scarce populations. Our results demonstrate that this one-pot RPA-Cas12a system provides a sensitive, specific, and practical solution for point-of-care TB diagnosis, offering a viable alternative to traditional laboratory-based methods.</p>

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A one-pot biplex RPA-Cas assay for sensitive detection of Mycobacterium tuberculosis from tongue swabs

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月27日

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  • Jason L. Cantera,
  • Mohammad Dehghan Banadaki,
  • Gayathri Nithiananthan,
  • Khushboo Khimani,
  • Julie L. Pendergraft,
  • Damian Madan,
  • Jamie Purcell,
  • John T. Connelly,
  • Eric A. Nalefski

摘要

The global management of tuberculosis (TB) is limited by the infrastructure requirements of current molecular diagnostics and the logistical burdens of sputum-based testing. While isothermal nucleic acid amplification provides a decentralized alternative, its clinical utility is often limited by amplicon carryover contamination and complex multi-step protocols. In this study we developed a fully integrated, “one-pot” biplex assay that couples recombinase polymerase amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‑associated protein Cas12a-mediated trans-cleavage within a single, homogeneous reaction. This single-pot chemistry that targets the IS1081 and IS6110 genes of Mycobacterium tuberculosis eliminates the need for post-amplification handling, addressing a primary failure mode of decentralized testing, while maintaining an analytical sensitivity of 1.1 genomes per 15-µL reaction. The assay delivers sample-to-result within 30 min, with no observed cross-reactivity with common nontuberculous mycobacteria and respiratory pathogens. We further validated the system using clinical tongue swab samples, a non-invasive sampling method recently recognized by the World Health Organization to improve diagnostic yield in sputum-scarce populations. Our results demonstrate that this one-pot RPA-Cas12a system provides a sensitive, specific, and practical solution for point-of-care TB diagnosis, offering a viable alternative to traditional laboratory-based methods.