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, Chaosu Hu, Huan Huang, Yang Liu, Guoping Zhao, Xiao‐Yong Fan, Shuihua Lu, Ye Hu, Jin Wang
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. The clinical adaptation of CRISPR assays is constrained by complexity and the absence of quality control. Here, authors report a thermally regulated asynchronous CRISPR-enhanced assay enabling direct, highly sensitive one-step detection of monkeypox virus and a human gene in clinical samples.