Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance

Zhen Huang, Yajuan Dong, Yang Yang, Xuchun Han, Fuxiang Wang, Christopher J. Lyon, Shuai Ding, Yun Peng, Ganggang Zhang, Christina Hu, Huan Huang, Liu Yang, Guoping Zhao, Xiao-Yong Fan (), Shuihua Lu (), Tony Hu () and Jin Wang ()
Additional contact information
Zhen Huang: Tulane University School of Medicine, Center for Cellular and Molecular Diagnostics
Yajuan Dong: Fudan University, Shanghai Public Health Clinical Center & Shanghai Institute of Infectious Diseases and Biosecurity
Yang Yang: Southern University of Science and Technology, Shenzhen Third People’s Hospital
Xuchun Han: Wuxi Tolo Biotechnology Co., Ltd.
Fuxiang Wang: Southern University of Science and Technology, Shenzhen Third People’s Hospital
Christopher J. Lyon: Tulane University School of Medicine, Center for Cellular and Molecular Diagnostics
Shuai Ding: Wuxi Tolo Biotechnology Co., Ltd.
Yun Peng: Southern University of Science and Technology, Shenzhen Third People’s Hospital
Ganggang Zhang: Jiangxi Academy of Sciences, Institute of Microbiology
Christina Hu: Tulane University School of Medicine, Center for Cellular and Molecular Diagnostics
Huan Huang: National Clinical Research Center for Infectious Diseases
Liu Yang: Southern University of Science and Technology, Shenzhen Third People’s Hospital
Guoping Zhao: University of Chinese Academy of Sciences, Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study
Xiao-Yong Fan: Fudan University, Shanghai Public Health Clinical Center & Shanghai Institute of Infectious Diseases and Biosecurity
Shuihua Lu: National Clinical Research Center for Infectious Diseases
Tony Hu: Tulane University School of Medicine, Center for Cellular and Molecular Diagnostics
Jin Wang: Wuxi Tolo Biotechnology Co., Ltd.

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: Abstract The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection – 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.

Date: 2025
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DOI: 10.1038/s41467-025-65193-1

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