Highly sensitive and specific detection of aminoglycoside resistance methylase gene armA by multiple cross displacement amplification-CRISPR/Cas12a technique

Introduction The 16S rRNA methylase armA serves as a key molecular determinant conferring high-level resistance to aminoglycoside antibiotics in bacterial pathogens. Development of a rapid and reliable armA gene detection assay will improve surveillance of enzyme-producing strains and support drug resistance control in diverse ecological settings. Methods A multiple cross displacement amplification (MCDA) combined with CRISPR/Cas12a system assay was established to identify armA gene with rapidity, specificity, and sensitivity. Results The entire experimental process, including genomic DNA extraction (15 minutes), MCDA reaction (40 minutes), CRISPR/Cas12a-mediated cleavage (5 minutes), and fluorescence detection (10 minutes), can be completed within 70 minutes. In terms of analytical specificity, the assay yielded positive results for all armA-positive isolates and negative results for all non- armA samples. The sensitivity of the MCDA-CRISPR/Cas12a assay reached a detection limit as low as 10 copies/mL of armA-positive plasmid DNA. In 129 cultured clinical isolates validated by whole-genome sequencing, the assay achieved 100% concordance for armA detection. Discussion The technique established in this study provides a rapid and accurate diagnostic approach for laboratory-based surveillance of armA in clinical and livestock settings, with future potential for point-of-care adaptation following further simplification and validation.