Universal Smartphone-Assisted Colorimetric Quantitative Platform Based on CRISPR Cas12a Detection for Sensitive and Rapid Diagnosis of Multiple Virulence Genes of Hypervirulent Klebsiella pneumoniae.

Over the past few decades, the prevalence of hypervirulent Klebsiella pneumoniae (hvKp) in the Asia-Pacific region, notably associated with community-acquired pyogenic liver abscesses, has been alarming. Most existing hvKp detection methods are time-consuming and rely on expensive equipment, which is particularly problematic in resource-limited settings. Thus, there is an urgent need for convenient, rapid, and cost-effective approaches to the identification and quantification of hvKp. In this study, we developed a CRISPR Cas12a-based smartphone-assisted colorimetric quantitative platform (Cas12a-SACQP), a portable platform enabling visible nucleic acid detection and analysis via a smartphone application with on-site detection capability. We applied Cas12a-SACQP to detect multiple virulence genes of hvKp, including rmpA, iroB, iucA, and peg-344. The limits of detection (LODs) for these genes were 2.42 CFU/mL for rmpA, 2.33 CFU/mL for iroB, 1.72 CFU/mL for iucA, and 2.83 CFU/mL for peg-344, respectively. We simultaneously tested Klebsiella pneumoniae clinical strains from various samples (ear discharge, sputum, and blood) for these virulence genes. Compared with conventional PCR identification and real-time PCR, Cas12a-SACQP effectively differentiates classical Klebsiella pneumoniae (cKp) from hvKp from these diverse clinical sources. Furthermore, the entire Cas12a-SACQP workflow can be performed using portable devices, and the results can be rapidly visualized via a smartphone. This platform, therefore, holds substantial potential for rapid and quantitative detection of clinical pathogens in resource-constrained environments.