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<p>Most CRISPR assays lack clinical utility due to their complex workflows and limited validation. Here we present a streamlined “one-pot” asymmetric CRISPR tuberculosis assay that attenuates amplicon degradation to achieve 5 copies/μL sensitivity within 60 min and detect positive patient samples within 15 min. This assay exhibited 93%, 83%, and 93% sensitivity with adult respiratory, pediatric stool, and adult cerebral spinal fluid specimens, and detected 64% of clinically diagnosed tuberculous meningitis cases, in a cohort of 603 clinical samples. This assay achieves complete specificity and greater sensitivity (74% vs. 56%) than the most sensitive reference test with prospectively collected tongue swabs, and exhibits similar performance when adapted to a lateral flow assay format and employed to analyze self-collected tongue swabs. These results demonstrate the utility of this approach across diverse specimen types, including those suitable for use in remote and resource-limited settings, to improve access to molecular diagnostics.</p>

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Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年9月17日

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  • Zhen Huang,
  • Zhe Song,
  • Jianfeng Zeng,
  • Xuhui Liu,
  • Mutong Fang,
  • Zhiyuan Wu,
  • Yao Zhao,
  • Yanli Chen,
  • Dan Li,
  • Huan Huang,
  • Liang Fu,
  • Peng Xu,
  • Bo Ning,
  • Jun Chen,
  • Ming Guan,
  • Lin Sun,
  • Christopher J. Lyon,
  • Xiao-Yong Fan,
  • Shuihua Lu,
  • Tony Hu

摘要

Most CRISPR assays lack clinical utility due to their complex workflows and limited validation. Here we present a streamlined “one-pot” asymmetric CRISPR tuberculosis assay that attenuates amplicon degradation to achieve 5 copies/μL sensitivity within 60 min and detect positive patient samples within 15 min. This assay exhibited 93%, 83%, and 93% sensitivity with adult respiratory, pediatric stool, and adult cerebral spinal fluid specimens, and detected 64% of clinically diagnosed tuberculous meningitis cases, in a cohort of 603 clinical samples. This assay achieves complete specificity and greater sensitivity (74% vs. 56%) than the most sensitive reference test with prospectively collected tongue swabs, and exhibits similar performance when adapted to a lateral flow assay format and employed to analyze self-collected tongue swabs. These results demonstrate the utility of this approach across diverse specimen types, including those suitable for use in remote and resource-limited settings, to improve access to molecular diagnostics.