为了解决CRISPR/Cas12f1系统在临床诊断中的局限性，需要单链DNA（ssDNA）或体外转录本（RNA）的复杂制备，我们开发了一种名为PDTCTR（PAM依赖的dsDNA靶标激活的Cas12f1TransReporter）的荧光生物传感器。这种创新的生物传感器集成了重组酶聚合酶扩增（RPA）与Cas12f_ge4.1系统，促进双链DNA（dsDNA）的直接检测。PDTCTR代表了一个重大的飞跃，表现出比原始Cas12f1系统高一百倍的检测灵敏度。它证明了检测肺炎支原体(M.肺炎）和乙型肝炎病毒（HBV）具有10个拷贝每微升（16.8aM）的出色敏感性，并以高精度区分单核苷酸变异（SNV），包括非小细胞肺癌（NSCLC）中普遍存在的EGFR（L858R）突变。PDTCTR的临床评估显示出其高灵敏度和特异性，费率从93%-100%到100%，分别,强调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CRISPR技术在临床诊断方面的重大进步及其在早期疾病检测和个性化医疗方面的广阔前景。
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.
