Song Shao-Zheng, Li Zi-Yuan, Liu Yuan-Yuan, Wu Ying-Chao, Yu Kang-Ying, He Zhengyi
School of Health and Nursing/Department of Basic, Wuxi Taihu University, Wuxi, Jiangsu, China.
School of Health and Nursing/Department of Nursing, Wuxi Taihu University, Wuxi, Jiangsu, China.
Front Cell Infect Microbiol. 2025 Jan 3;14:1493492. doi: 10.3389/fcimb.2024.1493492. eCollection 2024.
To establish a rapid detection method for canine using recombinase-aided amplification (RAA) technology.
The outer membrane protein 25 gene fragment (Omp25) of canis was targeted. Primers and fluorescent probes were designed and synthesized, and recombinant plasmids were constructed as standards. The RAA assay was optimized by screening primers and establishing a fluorescent reaction system. Sensitivity was analyzed using plasmid standards with varying copy numbers. Specificity was tested using genomes from canis, suis, melitensis, abortus, spp. Reproducibility was evaluated using plasmid standards from the same and different batches.
The optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39°C for 15 minutes. The detection sensitivity was 1 copy/μL. No cross-reaction was observed with other species or pathogenic bacteria, indicating high specificity. Intra-batch variability was below 1.00%, and inter-batch variability was below 2.00%. The positive detection coincidence rate of RAA was significantly higher than that of commercial real-time fluorescence quantitative PCR (100% VS 86.96%, P<0.05).
The RAA-based rapid detection method for canis is suitable for clinical rapid testing. It offers advantages such as quick detection, high sensitivity, strong specificity, and good reproducibility. This method provides new insights for the rapid detection of canine and the precise diagnosis of other pet diseases, making it suitable for promotion and application.
建立一种基于重组酶介导等温扩增（RAA）技术的犬种快速检测方法。
以犬种的外膜蛋白25基因片段（Omp25）为靶点。设计并合成引物和荧光探针，构建重组质粒作为标准品。通过筛选引物和建立荧光反应体系对RAA检测方法进行优化。使用不同拷贝数的质粒标准品分析灵敏度。使用犬种、猪种、羊种、牛种、其他种的基因组测试特异性。使用同一批次和不同批次的质粒标准品评估重复性。
优化后的RAA体系使用引物bOmp25-F2/bOmp25-R2和探针bOmp25-P，反应温度恒定为39°C，反应15分钟。检测灵敏度为1拷贝/μL。与其他种或病原菌未观察到交叉反应，表明特异性高。批内变异系数低于1.00%，批间变异系数低于2.00%。RAA的阳性检测符合率显著高于商业实时荧光定量PCR（100%对86.96%，P<0.05）。
基于RAA的犬种快速检测方法适用于临床快速检测。它具有检测快速、灵敏度高、特异性强、重复性好等优点。该方法为犬种的快速检测及其他宠物疾病的精准诊断提供了新的思路，适合推广应用。
