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

IF 15.7 1区综合性期刊 Q1 MULTIDISCIPLINARY SCIENCES

Nature Communications Pub Date : 2025-09-17 DOI:10.1038/s41467-025-63094-x

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

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引用次数: 0

Abstract

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.

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用多导RNA Cas12a检测敏感病原体DNA，有利于反式与顺式切割。

大多数CRISPR分析由于其复杂的工作流程和有限的验证而缺乏临床实用性。在这里，我们提出了一种流线型的“一锅”非对称CRISPR结核检测方法，该方法可以降低扩增子降解，在60分钟内达到5拷贝/μL的灵敏度，并在15分钟内检测到阳性患者样本。该方法对成人呼吸道、儿童粪便和成人脑脊液标本的敏感性分别为93%、83%和93%，并在603例临床样本中检测出64%的临床诊断的结核性脑膜炎病例。与最敏感的前瞻性舌拭子参考试验相比，该试验具有完全的特异性和更高的灵敏度（74%对56%），并且在适应横向流动试验格式并用于分析自采舌拭子时表现出相似的性能。这些结果证明了这种方法在不同标本类型上的实用性，包括那些适合在偏远和资源有限的环境中使用的标本，以改善分子诊断的可及性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Nature Communications Biological Science Disciplines-

CiteScore

24.90

自引率

2.40%

发文量

6928

审稿时长

3.7 months

期刊介绍： Nature Communications, an open-access journal, publishes high-quality research spanning all areas of the natural sciences. Papers featured in the journal showcase significant advances relevant to specialists in each respective field. With a 2-year impact factor of 16.6 (2022) and a median time of 8 days from submission to the first editorial decision, Nature Communications is committed to rapid dissemination of research findings. As a multidisciplinary journal, it welcomes contributions from biological, health, physical, chemical, Earth, social, mathematical, applied, and engineering sciences, aiming to highlight important breakthroughs within each domain.