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Journal of Integrative Agriculture  2025, Vol. 24 Issue (12): 4815-4820    DOI: 10.1016/j.jia.2025.05.023
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Development of recombinase-aided amplification assays with real-time fluorescence and lateral flow dipstick for the rapid detection of Actinobacillus pleuropneumoniae

Haoran Kang1, 2, Deyu Li1, 2, Cheng Song3, Yongning Zhang1, 2, Lei Zhou1, 2, Xinna Ge1, 2, Jun Han1, 2, Xin Guo1, 2#, Hanchun Yang1, 2

1 National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

2 Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

3 JOFUNHWA Biotechnology Co., Ltd., Beijing Biomedical Technology Center, Beijing 102600, China

 Highlights 
The recombinase-aided amplification (RAA) assays were developed for the rapid detection of Actinobacillus pleuropneumoniae.
The results not only can be detected by real-time fluorescence readout but also can be visualized by a portable blue light instrument and lateral flow dipsticks.
These assays provided reliable tools for preventing A. pleuropneumonia, especially in on-site diagnostics.
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摘要  

猪传染性胸膜肺炎PCP是由猪胸膜肺炎放线杆菌A. pleuropneumoniae引起的猪的高度传染性、接触性呼吸道疾病,严重制约着我国生猪产业持续健康的发展。早期的快速诊断和实时监测对于该病的防控至关重要。目前常用的诊断方法存在操作复杂、耗时长、对仪器设备要求较高等问题,难以满足临床上早期、快速诊断以及临床基层和现场检测的实际需求。本研究旨在建立快速、便捷、结果可视化的A. pleuropneumoniae重组酶介导的等温扩增(RAA)快速检测方法,以期为该病的防控提供技术手段。通过比对分析GenBank1~18血清型A. pleuropneumoniae apxIVA基因序列设计exo针和nfo探针并系统性筛选了RAA引物对,分别建立了A. pleuropneumoniae实时荧光和试纸条(LFDRAA快速检测方法。实时荧光RAA方法不仅可以通过荧光定量PCR仪进行检测(real-time RAA)还可以借助便捷式蓝光成像仪对结果进行肉眼可视化判定(visual RAA)。该方法42°C等温条件下30 min内即可完成扩增,反应快速、简单;该方法能够检测所有血清型A. pleuropneumoniae与其他常见的临床致病菌没有交叉反应,特异性好real-time RAARAA-LFD方法的检测敏感性17.9 copies/µL95%置信区间),visual RAA方法的检测敏感性203.5 copies/µL95%置信区间),敏感性强;通过对85份临床样品进行检测,real-time RAA方法与qPCR方法的检测符合率为97.6%RAA-LFDvisual RAA方法与qPCR方法的检测符合率为100%,临床检测性能好。本研究建立了A. pleuropneumoniae RAA快速检测方法,能够借助便捷式蓝光成像仪和LFD实现结果的可视化,特异性好、敏感性强、临床检测性能好,可作为临床基层和现场快速检测A. pleuropneumoniae可靠的诊断工具



Received: 14 December 2024   Accepted: 23 April 2025 Online: 27 May 2025  
Fund: 

This study was supported by the University-Industry Collaborative Education Program, China (220904860093831).

About author:  Haoran Kang, E-mail: 18260068857@163.com; #Correspondence Xin Guo, E-mail: guoxincau@cau.edu.cn

Cite this article: 

Haoran Kang, Deyu Li, Cheng Song, Yongning Zhang, Lei Zhou, Xinna Ge, Jun Han, Xin Guo, Hanchun Yang. 2025. Development of recombinase-aided amplification assays with real-time fluorescence and lateral flow dipstick for the rapid detection of Actinobacillus pleuropneumoniae. Journal of Integrative Agriculture, 24(12): 4815-4820.

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