The Affiliated Nanhua Hospital, Department of Clinical Laboratory, Hengyang Medical School, University of South China, Hengyang, China.
The Affiliated Nanhua Hospital, Department of Clinical Laboratory, Hengyang Medical School, University of South China, Hengyang, China; Department of Public Health Laboratory Sciences, College of Public Health, Hengyang Medical School, University of South China, Hengyang, China.
Biosens Bioelectron. 2024 Sep 15;260:116428. doi: 10.1016/j.bios.2024.116428. Epub 2024 May 24.
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.
为了解决 CRISPR/Cas12f1 系统在临床诊断中的局限性，该系统需要复杂的单链 DNA（ssDNA）或体外转录本（RNA）的制备，我们开发了一种名为 PDTCTR（PAM 依赖性 dsDNA 靶标激活 Cas12f1 转报告子）的荧光生物传感器。这种创新的生物传感器将重组酶聚合酶扩增（RPA）与 Cas12f_ge4.1 系统相结合，便于直接检测双链 DNA（dsDNA）。PDTCTR 是一个重大的突破，其检测灵敏度比原始 Cas12f1 系统高 100 倍。它能够以 10 拷贝/微升（16.8 aM）的优异灵敏度检测肺炎支原体（M. pneumoniae）和乙型肝炎病毒（HBV），并具有高精度区分单核苷酸变异（SNV）的能力，包括非小细胞肺癌（NSCLC）中常见的 EGFR（L858R）突变。PDTCTR 的临床评估表明其具有高灵敏度和特异性，其灵敏度和特异性分别为 93%-100%和 100%，这突显了其在传染病和癌症相关 SNV 诊断方法方面的潜力。这项研究强调了 CRISPR 技术在临床诊断方面的重大进展及其在早期疾病检测和个性化医疗方面的广阔前景。
