Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Rapid Detection of Ciprofloxacin-Resistance Mutations in Clinical Urinary Isolates

Abstract Fluoroquinolone resistance among uropathogens is rising worldwide, yet culture-based susceptibility testing is time-consuming, and molecular reference methods remain instrument-dependent. We developed an isothermal assay coupling recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans -cleavage to identify the chromosomal quinolone-resistance-determining region (QRDR) mutations underlying ciprofloxacin (CIP) resistance in Escherichia coli , Klebsiella pneumoniae and Proteus mirabilis . Sanger sequencing of gyrA , parC and parE in 137 clinical isolates (58 CIP-resistant, 79 CIP-susceptible) identified GyrA S83L/D87N ( E. coli ), GyrA S83I ( K. pneumoniae and P. mirabilis ), ParC S80I and ParE S458A as the predominant substitutions. Seven CRISPR RNAs (crRNAs) interrogated five RPA amplicons. Against sequencing-confirmed genotypes, individual crRNAs achieved sensitivities of 60–100% and specificities of 77–100% by RPA, comparable to PCR-CRISPR. Every cross-reaction arose in an isolate carrying GyrA S83L, which creates the motif Cas12a must bind before cutting; no wild-type gyrA was called positive. Against phenotype, a combined E. coli panel flagged all 18 resistant isolates (100% sensitivity, 75% specificity), a combined K. pneumoniae ParC panel reached 58% sensitivity, and the P. mirabilis GyrA assay 95% sensitivity, both at 100% specificity. From cultured isolates, the workflow returns a visual result within one hour using only a constant heat source, well ahead of phenotypic testing.