Rapid and simultaneous detection of <i>Escherichia coli and Klebsiella pneumoniae:</i> a novel dual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats-Cas12a method

<i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> are major hospital-acquired pathogens, posing severe threats to critically ill and immunocompromised patients. Their pathogenicity and virulence factors easily cause invasive infections, endangering patient health and life. This study developed a dual recombinase polymerase amplification combined with Clustered Regularly Interspaced Short Palindromic Repeats-Cas12a assay for rapid, simultaneous, specific detection of <i>E. coli uidA</i> and <i>K. pneumoniae rcsA</i> genes. The assay operated at 37°C, and the total reaction time was approximately 70 min. The analytical sensitivity for <i>E. coli</i> and <i>K. pneumoniae</i> was 5.37 × 10¹ copies/μL and 5.90 × 10¹ copies/μL, respectively. It showed excellent specificity (no cross-reactivity) and 100% concordance with PCR results in clinical sample validation. This assay overcame limitations of traditional methods, such as bacterial culture (time-consuming) and PCR (the dependency on expensive thermal cyclers), and offers advantages of rapidity, high sensitivity, simplicity, and cost-effectiveness, providing a powerful tool for rapid, accurate clinical diagnosis of <i>E. coli</i> and <i>K. pneumoniae</i> infections.IMPORTANCE<i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> are the main pathogens causing hospital-acquired infections, which can lead to serious complications and pose a significant challenge to public health. Therefore, establishing rapid, sensitive, specific, and reliable detection methods for <i>E. coli</i> and <i>K. pneumoniae</i> is of great significance for promoting accurate early clinical diagnosis and guiding treatment decisions. With the increasing incidence of mixed infections, single-target nucleic acid testing can no longer meet clinical needs. In this study, the efficient recombinase polymerase amplification (RPA) isothermal amplification technique was combined with the highly sensitive Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a detection system to successfully develop a duplex RPA-CRISPR-Cas12a method. This system can specifically and simultaneously identify <i>E. coli</i> (targeting the <i>uidA</i> gene) and <i>K. pneumoniae</i> (targeting the <i>rcsA</i> gene) in a single detection process.