Establishment and application of recombinase polymerase amplification combined with a lateral flow dipstick for the detection of mcr-1 in uncultured clinical samples

The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacterial which suffer from complexity, time-consuming process, and skilled personnel requirements, making them unsuitable for field analysis. A rapid detection technique combining recombinase polymerase amplification (RPA) with lateral flow dipstick (LFD) targeting on uncultured clinical samples was developed. This new method targeting the mcr-1 gene region (23,232-23,642 bp, No. KP347127.1), achieved a low detection limit of 10 copies/μL. The whole process was carried out with high specificity and completed within 20 minutes. Evaluation assay was conducted using 45 human fecal samples and 16 strains yielded an 98% accuracy, which are closely matching antimicrobial susceptibility outcomes. The novel method integrates nucleic acid extraction, isothermal amplification, and test assay, suggest the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts.