【摘要】 猪肺炎支原体（Mhp）是引起猪支原体肺炎（MPS）的病原，给全球养猪业带来了巨大的经济损失。早期快速诊断和实时监测对该病的防控至关重要，目前常用的核酸检测方法难以满足临床上即时检测（POCT）的实际需求。本试验针对Mhp细胞膜表面脂蛋白基因p46，通过序列比对和系统性的引物筛选设计了exo探针及其对应的重组酶介导的等温扩增（RAA）技术引物对，建立了Mhp实时荧光（real-time）RAA快速检测方法；随后，对该方法的特异性、敏感性和重复性进行检测，并使用该方法和实时荧光定量PCR(qPCR)对比检测临床样本。结果显示，该方法与猪链球菌、肺炎克雷伯氏菌、猪格拉瑟氏菌、猪胸膜肺炎放线杆菌、金黄色葡萄球菌、铜绿假单胞菌、鼠伤寒沙门菌和大肠杆菌均无交叉反应，特异性好；Mhp real-time RAA及其可视化方法的检测敏感性分别为18和29 copies/μL(95%置信区间)，敏感性强；组内和组间重复性试验变异系数均小于8.0%，重复性好；对108份猪临床样本的检测结果显示，该方法及其可视化方法与Mhp qPCR方法的符合率分别为98.15%和99.07%。本试验所建立Mhp real-time RAA方法可以通过便捷式蓝光仪器实现结果的可视化，更适用于资源匮乏的诊断实验室和基层现场，为MPS的防控提供了技术手段。更多还原

【Abstract】 Mycoplasma hyopneumonia(Mhp) is the pathogen causing swine mycoplasmal pneumonia(MPS), which leads to significant economic losses in the global pig industry. Early rapid diagnosis and real-time monitoring are crucial for the prevention and control of this disease. However, commonly used nucleic acid detection methods are inadequate for point-of-care testing(POCT) in clinical practice. In this study, primers targeting the Mhp cell membrane surface lipoprotein gene p46 were designed through sequence alignment and systematic primer screening, and a corresponding real-time recombinase-aided amplification(RAA) method was established for rapid Mhp detection. The specificity, sensitivity, and repeatability of the method were evaluated, and clinical samples were tested using this method in comparison with quantitative real-time PCR(qPCR). The results showed that the method exhibited no cross-reactivity with Streptococcus suis, Klebsiella pneumoniae, Glaesserella parasuis,Actinobacillus pleuropneumonia, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, and Escherichia coli, demonstrating excellent specificity. The sensitivity of the Mhp real-time RAA and its visualization method was 18 and 29 copies/μL(95% confidence interval), respectively, showing strong sensitivity. The intra-and inter-assay coefficients of variation were both less than 8. 0%, indicating good repeatability. Testing of 108 clinical pig samples showed that the concordance rates of this method and its visualization method with Mhp qPCR were 98. 15% and 99. 07%, respectively. The Mhp real-time RAA method established in this study can achieve result visualization using portable blue light instruments, making it more suitable for diagnostic laboratories with limited resources and grassroots field settings. It provides a technological means for the prevention and control of MPS.更多还原