A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection

Abstract

Rapid and accurate detection of viable bacteria is essential for the clinical diagnosis of urinary tract infections (UTIs) and for making effective therapeutic decisions. However, most current molecular diagnostic techniques are unable to differentiate between viable and non-viable bacteria. In this study, we introduce a novel isothermal platform that integrates strand exchange amplification (SEA) with the CRISPR/Cas12a system, thereby enhancing both the sensitivity and specificity of the assay and achieving detection of phage DNA at concentrations as low as 4 × 10² copies per μL. Moreover, the incorporation of phages facilitates the specific recognition of viable bacteria and amplifies the initial signal through the inherent specificity and propagation properties of these phages. By employing the phage-assisted SEA-Cas12a approach, we successfully detected viable bacteria in human urine samples without the necessity of DNA extraction within 3.5 hours, achieving a detection limit of 10³ CFU per mL. Considering its speed, accuracy, and independence from specialized equipment, this platform demonstrates significant potential as a robust tool for the rapid detection of various pathogens in resource-limited settings, thereby facilitating timely clinical management of UTI patients.