Streptococcus equi subspecies equi (S. equi) is the causative pathogen of strangles in horses, donkeys, and other equine animals. Strangles has spread globally and causes significant losses to the horse industry. In response to the urgent need for effective disease control, this study introduces a novel nucleic acid diagnostic method known as a real-time recombinase-assisted amplification (RAA) assay, developed based on the eqbE gene, for the rapid detection of S. equi nucleic acid. The real-time RAA method employs specifically designed probes and primers targeting the eqbE gene, enhancing the overall specificity and sensitivity of the detection. After efficiency optimization, this real-time RAA method can detect 10 or more copies of nucleic acid within 20 min. The method demonstrates high specificity for S. equi and does not cross-react with other clinically relevant pathogens. Real-time RAA diagnostic performance was evaluated using 98 nasal swab samples collected from horses and compared with the real-time PCR detection method. Results revealed that 64 and 65 samples tested positive for S. equi using real-time RAA and real-time PCR, respectively. The overall agreement between the two assays was 96.94% (95/98), with a kappa value of 0.931 (p < 0.001). Further linear regression analysis indicated a significant correlation in the detection results between the two methods (R² = 0.9012, p < 0.0001), suggesting that the real-time RAA assay exhibits a detection performance comparable to that of real-time PCR. In conclusion, the real-time RAA assay developed here serves as a highly specific and reliable diagnostic tool for the detection of S. equi in equine samples, offering a potential alternative to real-time PCR methods. In conclusion, the real-time RAA nucleic acid diagnostic method, based on the eqbE gene, offers rapid and accurate diagnosis of S. equi, with the added advantage of minimal equipment requirements, thus contributing to the efficient detection of strangles in horses.

Strangles, caused by Streptococcus equi subsp. equi (S. equi), is a highly contagious respiratory disease that causes substantial economic losses to the horse industry worldwide. Serological diagnosis currently relies mainly on the imported IDvet^® dual-antigen sandwich ELISA kit, which shows good diagnostic performance but is expensive and lacks a domestic alternative in China, thereby limiting its routine use in primary veterinary settings. The aim of this study was to develop a domestic indirect ELISA based on novel antigen targets and an untagged antigen purification strategy. Using comparative bioinformatics analyses, two conserved fragments with species-level specificity for S. equi, designated EQ8 (150 aa) and SclF (44 aa), were identified and fused for recombinant expression. The fusion protein was purified by GST affinity chromatography, cleaved with PreScission protease, and subjected to secondary purification to obtain an untagged EQ8–SclF protein. Western blotting and ELISA demonstrated that the purified protein retained good immunoreactivity and that the GST tag did not cause detectable cross-reactivity, with all OD₄₅₀ values obtained using GST-coated plates remaining below 0.09, well under the cut-off value of 0.115. Checkerboard titration established the optimal assay conditions as an antigen coating concentration of 1 μg/mL and a serum dilution of 1:100. Using the IDvet kit as the reference method, 157 clinical serum samples (67 positive and 90 negative) were tested in parallel. The newly developed assay showed a relative sensitivity of 89.55% (60/67), a relative specificity of 94.44% (85/90), an overall agreement rate of 92.36% (145/157), and a kappa value of 0.84, indicating excellent agreement between the two methods. Good inter-assay reproducibility was observed, with coefficients of variation all below 9.29%. In conclusion, an indirect ELISA based on an untagged EQ8–SclF fusion protein was preliminarily established for the serological detection of strangles. This assay showed diagnostic performance highly consistent with that of the imported commercial kit while offering potential advantages in terms of lower cost, simpler operation, and elimination of tag-associated interference. It therefore shows potential as a domestic alternative for the serological diagnosis of strangles.