Rapid detection of Klebsiella pneumoniae based on one-tube RPA-CRISPR/Cas12a system

Klebsiella pneumoniae (KP) is a prevalent pathogen implicated in both community-acquired and nosocomial infections, often leading to severe clinical outcomes. The conventional methods for KP identification are characterized by intricacy and suboptimal efficiency. In this research, we have engineered a novel One-Tube RPA- CRISPR/Cas12a system, integrating recombinase polymerase amplification (RPA) method with the CRISPR/Cas12a diagnostic platform, to facilitate the detection of K. pneumoniae. To minimize the likelihood of aerosol-based contamination, the RPA components are positioned at the base of the tube, while the CRISPR/Cas12a components are placed at the tube's cap. The systems are combined post-RPA amplification through a brief centrifugation step, ensuring that RPA reactions are conducted independently to produce an adequate amount of target DNA before interaction with the CRISPR/Cas12a system. This method was validated using both fluorescent and lateral flow strip assays, achieving a limit of detection (LOD) of 10⁰ copies/μL and 10¹ copies/μL respectively. The specificity for KP detection was found to be 100 %. Furthermore, the system demonstrated a positivity rate of 78 % (18/23) when directly extracting DNA from sputum samples, corroborated by culture and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The simplicity and rapidity of the assay are augmented by a straightforward sample processing without extraction. The complete assay duration from specimen receipt to result is approximately 40 min, significantly reducing the turnaround time (TAT). Collectively, this system presents a streamlined, expeditious, and highly specific diagnostic approach for the detection of Klebsiella pneumoniae strains.