Development of multiplex recombinase polymerase amplification for the rapid detection of five carbapenemase (bla_KPC, bla_NDM, bla_OXA-48-like, bla_IMP, and bla_VIM) and 10 mcr (mcr-1 to mcr-10) genes in blood cultures

The emergence of plasmid-encoded carbapenemase and mobile colistin resistance (mcr) genes poses a significant challenge in controlling the spread of multidrug-resistant Gram-negative bacteria. Addressing this issue requires the development of rapid, accurate, and cost-effective tools for gene detection. For the first time, this study reports three multiplex recombinase polymerase amplification (RPA) assays, each designed to detect five resistance genes: carbapenemase (bla_KPC, bla_NDM, bla_OXA-48-like, bla_IMP, and bla_VIM), mcr-1 to mcr-5, and mcr-6 to mcr-10. Using agarose gel electrophoresis, all 15 target genes were successfully amplified by the three assays, demonstrating the potential of these assays for integration with rapid reporting platforms. To increase their applicability, the assays were combined with SYBR^Ⓡ Green I for visual identification of all 15 target genes and with lateral flow immunoassays (LFIAs) for detection of two carbapenemase (bla_NDM and bla_OXA-48-like) and two mcr genes (mcr-1 and mcr-3) genes. Specificity testing showed that RPA-SYBR^Ⓡ Green I and RPA-LFIAs produced no cross-reactivity among the target genes. The limit of detection for RPA-SYBR^Ⓡ Green I, for all genes, ranged from 2 × 10⁰ to 2 × 10² CFU/reaction, and for RPA-LFIAs from 2 × 10⁰ to 2 × 10³ CFU/reaction. The developed RPA-SYBR^Ⓡ Green I and RPA-LFIAs successfully detected 15 and four target genes, from positive haemoculture bottles. These assays offer a promising approach for point-of-care testing. Providing a valuable tool for antimicrobial resistance surveillance and timely guidance for effective antibiotic intervention.