Construction And Application Of Novel Nucleic Acid Detection Systems With CRISPR-Cas

The fast,reliable and ultrasensitive detection of specific nucleic acid sequences has become increasingly important in many fields,such as food safety,clinical analysis,global biosecurity and biocontainment,as well as environmental monitoring.However,current methods for nucleic acid detection suffer from some limitations,such as low detection sensitivity,costly equipment,time-consuming methods,and false positive signal,which cannot meet the requirements of practical applications.Hence,the development of more efficient and low-cost nucleic acid detection methods is urgently needed.In recent years,beyond their widespread application as genome-editing and regulatory tools,CRISPR-Cas systems also play a critical role in nucleic acid detection due to their high sensitivity and specificity.In this study,a novel nucleic acid signal transduction and readout module was constructed with CRISPR-Cas12 a system and quenched fluorescent ss DNA reporters.Then several novel nucleic acid detection systems were developed through the ingenious combination of Cas12a-based nucleic acid signal transduction and readout module with EXPAR/PCR/RPA-based nucleic acid signal recognition module.Through which as low as 83 CFU/m L Salmonella typhimurium can be detected in complex environments within 1 h at room temperature.As the first study applying CRISPR-Cas12 a in miRNA detection and foodborne pathogen detection,these novel nucleic acid detection systems show promising prospect in the real-life applications of disease diagnosis and food analysis.