Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers.

Antimicrobial resistance (AMR) is a global public health problem particularly accentuated in low- and middle-income countries, largely due to a lack of access to sanitation and hygiene, lack of awareness and knowledge, and the inadequacy of molecular laboratories for timely and accurate surveillance programs. This study introduces a versatile molecular detection toolbox (C12a) for antibiotic resistance gene markers using CRISPR/Cas12a coupled to PCR. Our toolbox can detect less than 3×10 ng of DNA (100 attoMolar) or 10 CFU/mL. High concordance was observed when comparing the C12a toolbox with sequenced genomes and antibiotic susceptibility tests for the and antibiotic resistance genes (ARGs), which confer resistance to cefotaxime and other β-lactams, and amphenicols, respectively. C12a, designed to detect the Integrase 1 gene, confirmed a high prevalence of the integrase/integron system in containing multiple ARGs. The C12a toolbox was tested across a wide range of laboratory infrastructure including a portable setup. When combined with lateral flow assays (LFA), C12a exhibited competitive performance, making it a promising solution for on-site ARG detection. Altogether, this work presents a collection of molecular tools (primers, crRNAs, probes) and validated assays for rapid, versatile, and portable detection of antibiotic resistance markers, highlighting the C12a toolbox potential for applications in surveillance and ARG identification in clinical and environmental settings.