为了解决临床诊断中 CRISPR/Cas12f1 系统需要复杂的单链 DNA (ssDNA) 或体外转录本 (RNA) 制备的局限性，我们开发了一种名为 PDTCTR（PAM 依赖的 dsDNA 靶标激活）的荧光生物传感器。 Cas12f1 反式记者）。这种创新的生物传感器将重组酶聚合酶扩增 (RPA) 与 Cas12f_ge4.1 系统集成，有助于直接检测双链 DNA (dsDNA)。 PDTCTR 代表了一次重大飞跃，其检测灵敏度比原始 Cas12f1 系统高出一百倍。它展示了检测肺炎支原体 (M.pneumoniae) 和乙型肝炎病毒 (HBV) 的能力，灵敏度高达 10 个拷贝/微升 (16.8 aM)，并以高精度区分单核苷酸变异 (SNV)，包括 EGFR (L858R)非小细胞肺癌（NSCLC）中普遍存在的突变。 PDTCTR 的临床评估证明了其高敏感性和特异性，准确率分别为 93%-100% 和 100%，突显了其彻底改变传染病和癌症相关 SNV 诊断方法的潜力。这项研究强调了在用于临床诊断的 CRISPR 技术及其在早期疾病检测和个性化医疗方面的广阔前景。版权所有 © 2024。由 Elsevier B.V. 出版。

To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%-100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine.Copyright © 2024. Published by Elsevier B.V.