A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae (K. pneumoniae) was a prevalent nosocomial pathogen and a major cause of neonatal sepsis, highlighting the critical need for simple, rapid, sensitive and reliable diagnosis for K. pneumoniae infection. While CRISPR-based diagnostics provide a transformative technology to nucleic acid detection, current CRISPR-based methods often rely on PAM-dependent sequences, involve multiple steps, and are hampered by cumbersome manual operation, sequence limitations and the risk of aerosol contamination. To address these challenges, we developed a novel one-pot, one-step, PAM-independent CRISPR-Cas12b-based detection platform for rapid, sensitive and specific detection of K. pneumoniae, termed CRISPR-KP. This assay integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b system in a single reaction mixture, employing MCDA for target nucleic acid amplification and the trans-cleavage activity of CRISPR-Cas12b system for amplification signal decoding. This one-pot assay achieves a remarkable limit of detection 10 fg (∼1.6 copies) of K. pneumoniae DNA within a 40-min isothermal reaction at 59 °C, eliminating the need for complex multi-step procedures. CRISPR-KP demonstrated comparable diagnostic efficiency to the clinical standard method in bronchoalveolar lavage fluid (BALF) samples, with reduced turnaround time and minimal manual operation, highlighting the excellent reliability and accuracy of our assay. Taken together, the CRISPR-KP assay offers a promising diagnostic tool for K. pneumoniae detection in centralized laboratories, point-of-care settings, and primary healthcare institutions.