中国兽医杂志2025，Vol.61Issue(9)：1-9,9.DOI:10.20157/j.cnki.zgsyzz.2025.09.001
猪肺炎支原体实时荧光重组酶介导的等温扩增快速检测方法的建立和应用
Establishment and Application of a Real-Time Recombinase-Aided Amplification Rapid Detection Assay for Mycoplasma hyopneumoniae
摘要
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.关键词
重组酶介导的等温扩增技术/猪肺炎支原体/实时荧光定量PCR/快速检测Key words
recombinase-aided amplification/Mycoplasma hyopneumoniae/quantitative real-time polymerase chain reac-tion/rapid detection分类
农业科技引用本文复制引用
康浩然,杨汉春,黄雨薇,宋程,高鹏,张永宁,周磊,盖新娜,韩军,郭鑫..猪肺炎支原体实时荧光重组酶介导的等温扩增快速检测方法的建立和应用[J].中国兽医杂志,2025,61(9):1-9,9.基金项目
教育部产学合作协同育人项目(220904860093831) （220904860093831）
