Integration of multiplex PCR and CRISPR-Cas allows highly specific detection of multidrug-resistant Acinetobacter Baumannii

Bacterial identification and phenotypic antibiotic susceptibility test (p-AST) limit timely anti-infection treatment of patients in clinic to a large extent. Here, we develop a rapid platform integrated multiplex PCR with CRISPR-Cas array to detect multidrug-resistant Acinetobacter baumannii (MDRAB). The platform relies on multiplex PCR amplification strategy which can simultaneously amplify the house-keeping gene and 4 beta-lactamase genes of Acinetobacter baumannii (A. baumannii), accompanying with the indiscriminate single-stranded DNase activity of LbaCas12a to generate a single fluorescent signal for multiplex PCR products. The genotypic antibiotic susceptibility test (g-AST) of A. baumannii was completed within 2 h, with a detection limit down to 50 CFU/mL. In addition, we also proved the specificity to differentiate primer dimers. These findings jointly demonstrate the ultimate potential of the CRISPR-Cas12a method for multiple genes detection with high throughput and high specificity. Given the versatility and universality of CRISPR-Cas12a platform, it is expected that this research will further promote its application in the diagnosis and treatment of multidrug-resistant bacteria.