Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for Gene Detection of Colistin Resistant Isolates.

BACKGROUND/OBJECTIVES: The emergence of the mobilized colistin resistance 1 () gene, which causes colistin resistance, is a serious concern in animal husbandry, particularly in pigs. Although antibiotic regulations in many countries have prohibited the use of colistin in livestock, the persistence and dissemination of this plasmid-mediated gene require effective and rapid monitoring. Therefore, a rapid, sensitive, and specific method combining recombinase polymerase amplification (RPA) with an in-house lateral flow assay (LFA) for the gene detection was developed.

METHODS

The colistin agar test and broth microdilution were employed to screen 152 isolates from pig fecal samples of five antibiotic-used farms. The established RPA-in-house LFA was validated with PCR for gene detection.

RESULTS

The RPA-in-house LFA was completed within 35 min (20 min of amplification and 5-15 min on LFA detection) at 37 °C. The sensitivity, specificity, and accuracy were entirely 100% in concordance with PCR results. No cross-reactivity was detected with seven common pathogenic bacteria or other gene variants.

CONCLUSIONS

Therefore, the in-house RPA-LFA serves as a point-of-care testing tool that is rapid, simple, and portable, facilitating effective surveillance of colistin resistance in both veterinary and clinical settings, thereby enhancing health outcomes.