Real-time fluorescent recombinase-aided amplification for rapid detection of the multiple resistance gene optrA in Enterococcaceae pathogens

Abstract

The multiple resistance gene optrA is one of the primary causes of treatment failure when oxazolidinone antibiotics are used against infections caused by bacteria harboring this resistance gene. Globally, the prevalence of the multiple resistance gene optrA has been increasingly reported in food of animal origin, human populations, as well as along the entire food chain and associated environments. Notably, Enterococcaceae pathogens are recognized as the most important reservoir for such antibiotic resistance genes (ARGs). Therefore, there is an urgent need for a rapid, efficient, and accurate detection method to simultaneously screen and monitor such resistance genes in Enterococcaceae pathogens. In the present study, a simple, rapid, and visual real-time fluorescent recombinase-aided amplification (RT-RAA) assay was established for the specific detection of the multiple resistance gene optrA in Enterococcaceae pathogens. Primers and probe were designed based on the conserved region of optrA . A real-time fluorescent recombinase-aided amplification (RT-RAA) assay was developed. After optimizing reaction conditions, the assay was characterized for specificity, reproducibility and sensitivity using recombinant plasmids and bacterial isolates. A total of 100 retail chicken-derived Enterococcaceae isolates were subjected to parallel testing by RT-RAA, conventional PCR and qPCR to evaluate practical performance. This assay can complete the entire workflow, including crude DNA extraction, amplification, and result interpretation, within 40 min at 37 °C. Furthermore, the optimized fluorescent RAA assay can be initiated at body temperature, eliminating the requirement for precision instrumentation. Methodological validation demonstrated that the fluorescent RAA detection system exhibited high specificity, with no cross-reactivity observed with strains carrying other common resistance genes. Additionally, the assay showed good reproducibility and high sensitivity, with a minimum detection limit of 2.22 × 10¹ copies/µL. To further evaluate its clinical applicability, three detection methods—RAA, polymerase chain reaction (PCR), and real-time fluorescence quantitative PCR (qPCR)—were employed for parallel testing of 100 clinical chicken meat samples. The results indicated that the detection coincidence rate between RAA and PCR was 98%, while the coincidence rate between RAA and qPCR was 99%. The developed fluorescent RAA assay can effectively detect the multidrug resistance gene optrA in foodborne pathogens and holds great potential for on-site detection applications in clinical and food safety surveillance settings.

The authors' abstract, as published at the source. BMC Veterinary Research, 2026 · DOI ↗