Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales

The worldwide spread of carbapenemase-producing <i>Enterobacterales</i> (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (<i>bla</i>_KPC and <i>bla</i>_NDM) within 50 min, including 10 min for DNA extraction and 30-40 min for RCCS reaction (a 20 min RPA reaction with a 10-20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting <i>bla</i>_KPC and <i>bla</i>_NDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of <i>Klebsiella pneumoniae</i> and <i>Escherichia coli</i>; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.