Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi

Haoyu Zu; Rongkuan Sun; Jiaxin Li; Xing Guo; Min Wang; Wei Guo; Xiaojun Wang

doi:10.3390/microorganisms12040777

Development of a Real-Time Recombinase-Aided Amplification Method for the Rapid Detection of Streptococcus equi subsp. equi

Microorganisms. 2024 Apr 11;12(4):777. doi: 10.3390/microorganisms12040777.

Authors

Haoyu Zu¹, Rongkuan Sun^{1

2}, Jiaxin Li¹, Xing Guo¹, Min Wang¹, Wei Guo^{1

3}, Xiaojun Wang^{1

3}

Affiliations

¹ State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
² College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
³ Institute of Western Agriculture, The Chinese Academy of Agricultural Sciences, Changji 831100, China.

Abstract

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.

Keywords: Streptococcus equi subspecies equi; real-time RAA; strangles.

Grants and funding

This study was supported by the National Key Research and Development Project of China (grants number 2020YFE0203400) and The Natural Science Foundation of Heilongjiang Province (grant number TD2022C006).