Research Progress on Pathogen Detection Based on Integration of CRISPR-Cas12a and Isothermal Amplification Technology

Rapid and accurate detection of pathogenic bacteria is of great significance for public health security, disease control, and food safety assurance. In recent years, the clustered regularly interspaced short palindromic repeats-associated proteins 12a (CRISPR-Cas12a) system has emerged as a key tool for pathogenic bacteria detection due to its specific recognition via cis-cleavage and signal amplification capability through trans-cleavage. However, its application is restricted by challenges such as difficulty in detecting low-abundance targets and dependence on protospacer adjacent motif (PAM) sequences. Isothermal amplification technology can rapidly amplify target nucleic acids at a constant temperature, forming a complement to CRISPR-Cas12a. Combining CRISPR-Cas12a with isothermal amplification technology can effectively improve the sensitivity, specificity, and operational convenience of detection, making it suitable for POCT requirements. In this review, the molecular recognition and cleavage mechanisms of Cas12a were firstly outlined, and the characteristics and compatibility of various isothermal amplification methods were summarized. And the recent advances in their combined applications for pathogenic bacteria detection were reviewd, while the current limitations were discussed. Finally, the perspectives on future directions to guide further development in this field was provided.