ABSTRACT
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.
Subject(s)
Biosensing Techniques , CRISPR-Cas Systems , RNA, Guide, CRISPR-Cas Systems , Biosensing Techniques/methods , Humans , RNA, Guide, CRISPR-Cas Systems/genetics , Hepatitis B virus/genetics , Hepatitis B virus/isolation & purification , DNA/genetics , DNA/chemistry , Mycoplasma pneumoniae/genetics , Mycoplasma pneumoniae/isolation & purification , CRISPR-Associated Proteins/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/diagnosis , Bacterial Proteins/genetics , Bacterial Proteins/chemistry , Lung Neoplasms/genetics , Lung Neoplasms/diagnosis , Endodeoxyribonucleases/genetics , Endodeoxyribonucleases/chemistry , Pneumonia, Mycoplasma/diagnosisABSTRACT
Mycoplasma pneumoniae can cause severe respiratory tract infections and extrapulmonary diseases, which pose a significant threat to the health of children. Diagnostic methods for M. pneumoniae include isolation and culture, antibody detection, fluorescence quantitative PCR, and so on, but there are various shortcomings in time, cost, convenience, and sensitivity. In this study, we developed a rapid, sensitive, specific, and economical method for the detection of M. pneumoniae, termed the ERA/CRISPR-Cas12a dual system. The system used the high specificity and collateral cleavage activity of the LbCas12a protein, combined with enzymatic recombination amplification (ERA) technology with strong amplification ability, allowing the results to be observed by a portable fluorometer or visualized by the naked eye with a dipstick, which could be obtained in approximately 30 min. The ERA/CRISPR-Cas12a fluorescence and dipstick system were able to detect M. pneumoniae at titers as low as 1 and 100 copies/µL, respectively. The specificity of the two interpretation methods was 100%, and no cross-reaction with other pathogens was observed. In the evaluation of 92 clinical samples, the positive predictive agreements of the ERA/CRISPR-Cas12a fluorescence and dipstick systems with qPCR detection were 100% and 92.86%, respectively. The negative predictive agreements of both methods were 100%. In conclusion, this study established a portable, rapid, low-cost, ultrasensitive, and specific method for the early and rapid diagnosis of M. pneumoniae to meet the needs of on-site rapid detection in primary health institutions.
