Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for mcr-1 Gene Detection of Colistin Resistant Escherichia coli Isolates

• Published in: Antibiotics (Basel) Vol. 13; no. 10; p. 984
• Main Authors: Ullah, Naeem, Suchanta, Nutchaba, Pimpitak, Umaporn, Santanirand, Pitak, Am-In, Nutthee, Chaichanawongsaroj, Nuntaree
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 17.10.2024; MDPI
• Subjects: Animal culture; Animal husbandry; Antibiotics; Antibodies; Bacteria; Biological assay; Colistin; Cross-reactivity; E coli; Escherichia coli; Feces; Flow resistance; Gene flow; Genes; Genetic aspects; Gram-negative bacteria; lateral flow assay; Livestock; Methods; mobile colistin resistance 1 (mcr-1) gene; Physiological aspects; point-of-care testing; Polymerase chain reaction; Recombinase; recombinase polymerase amplification; Recombinases; Swine; Thailand; United Kingdom; Israel; point-of-care testing; recombinase polymerase amplification; lateral flow assay; Escherichia coli; mobile colistin resistance 1 (mcr-1) gene
• ISSN: 2079-6382; 2079-6382
• DOI: 10.3390/antibiotics13100984

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. 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. 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. 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.