Rapid 40-minute fluorescent test detects optrA resistance gene in Enterococcaceae

Researchers have developed a real-time fluorescent recombinase-aided amplification (RT-RAA) assay that can detect the multiple resistance gene optrA in Enterococcaceae pathogens within 40 minutes, running entirely at 37 °C without the need for precision laboratory instrumentation. The assay, described in BMC Veterinary Research, was designed to address the growing spread of oxazolidinone resistance genes along the food chain, where Enterococcaceae are recognized as the most important reservoir for such antibiotic resistance genes.

The optrA gene is one of the primary causes of treatment failure when oxazolidinone antibiotics are used against infections caused by bacteria harboring this resistance gene. Its prevalence has been increasingly reported globally in food of animal origin, in human populations, and along the entire food chain and associated environments. Because conventional detection approaches can be slow or equipment-dependent, the study’s authors set out to build a rapid, efficient, and accurate method capable of simultaneously screening and monitoring this resistance gene in Enterococcaceae pathogens.

To construct the assay, the team designed primers and a probe based on the conserved region of optrA. After optimizing the reaction conditions, they characterized the system for specificity, reproducibility, and sensitivity using recombinant plasmids and bacterial isolates. The resulting workflow is deliberately simple and visual: crude DNA extraction, amplification, and result interpretation can all be completed within the 40-minute window at body temperature, meaning the reaction can be initiated without a thermal cycler or other specialized equipment.

Methodological validation showed that the fluorescent RAA detection system exhibited high specificity, with no cross-reactivity observed against strains carrying other common resistance genes. The assay also demonstrated good reproducibility and high sensitivity, reaching a minimum detection limit of 2.22 × 10¹ copies per microliter. These performance characteristics were established using recombinant plasmids and bacterial isolates under controlled laboratory conditions.

To evaluate practical performance, the researchers subjected 100 retail chicken-derived Enterococcaceae isolates to parallel testing by RT-RAA, conventional PCR, and quantitative PCR (qPCR). The comparison indicated a detection coincidence rate of 98 percent between RAA and conventional PCR, and 99 percent between RAA and qPCR. The study describes these samples as clinical chicken meat samples, and the near-complete agreement between the three methods suggests the new assay performs comparably to established molecular techniques on real retail poultry material.

The authors conclude that the developed fluorescent RAA assay can effectively detect the multidrug resistance gene optrA in foodborne pathogens and holds potential for on-site detection applications in clinical and food safety surveillance settings. Because the reaction runs at body temperature and requires no precision instrumentation, the approach could in principle be deployed outside well-equipped laboratories, although the published evaluation was carried out with retail chicken-derived isolates rather than across broader sample types or geographic regions.

The significance of the target gene lies in its clinical impact. Oxazolidinones are a class of antibiotics whose effectiveness can be undermined when bacteria carry optrA, and the gene’s documented presence in animal-derived foods and food-chain environments makes rapid surveillance a public health priority. Enterococcaceae, as the recognized primary reservoir for such resistance genes, serve as a key sentinel group for monitoring this threat, which is why the study focused its assay specifically on this bacterial family.

As with any newly described diagnostic method, the reported findings reflect the specific validation performed in this study: specificity, reproducibility, and sensitivity testing on plasmids and isolates, followed by a 100-sample parallel comparison against PCR and qPCR. The detection limit of 2.22 × 10¹ copies per microliter and the high coincidence rates with reference methods define the assay’s demonstrated performance, while broader field deployment would depend on further evaluation beyond what this study reports.

Subject of Research: A rapid fluorescent recombinase-aided amplification assay for detecting the optrA antibiotic resistance gene in Enterococcaceae pathogens

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

Article References: Wang, F., Wang, Y.-X., Wang, S., Lian, K.-X., Zhu, Y.-K., Wang, Z.-X., Ding, X.-H., Wang, R.-N., Han, Y.-L., Yi, X., Cui, Y.-C., & Xuan, H.-Y. (2026). Real-time fluorescent recombinase-aided amplification for rapid detection of the multiple resistance gene optrA in Enterococcaceae pathogens. BMC Veterinary Research. https://doi.org/10.1186/s12917-026-06040-3

Image Credits: AI Generated

DOI: 10.1186/s12917-026-06040-3

Keywords: optrA, Enterococcaceae, antimicrobial resistance, recombinase-aided amplification, RT-RAA, oxazolidinones, food safety surveillance, rapid diagnostics, chicken meat isolates, qPCR comparison, point-of-need testing, BMC Veterinary Research