Development of recombinase-aided amplification assays with real-time fluorescence and lateral flow dipstick for the rapid detection of Actinobacillus pleuropneumoniae

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 Kang^{1, 2}, Deyu Li^{1, 2}, Cheng Song³, Yongning Zhang^{1, 2}, Lei Zhou^{1, 2}, Xinna Ge^{1, 2}, Jun Han^{1, 2}, Xin Guo^{1, 2}^#, Hanchun Yang^{1, 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

1. The recombinase-aided amplification (RAA) assays were developed for the rapid detection of A. pleuropneumoniae.

2. The results can not only be detected by real-time fluorescence readout but also can be visualized by a portable blue light instrument and lateral flow dipsticks.

3. These assays provided reliable tools for preventing A. pleuropneumonia, especially in on-site diagnostics.

Abstract
References

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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GenBank中1~18血清型A. pleuropneumoniae apxIVA基因序列设计了exo探针和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（LFD）RAA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）。该方法在42°C等温条件下30 min内即可完成扩增，反应快速、简单；该方法能够检测到所有血清型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 RAA和RAA-LFD方法的检测敏感性为17.9 copies/µL（95%置信区间），visual RAA方法的检测敏感性为203.5 copies/µL（95%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-LFD和visual 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。

Abstract

Porcine Contagious Pleuropneumonia (PCP) is an important bacterial infectious disease caused by Actinobacillus pleuropneumoniae (A. pleuropneumoniae), which has caused serious economic losses to the pig industry. Early rapid diagnosis and pathogen surveillance are essential for the prevention of this disease. In this study, recombinase-aided amplification (RAA) assays with real-time fluorescence and lateral flow dipsticks (LFD) for the rapid detection of A. pleuropneumoniae were established, optimized, and evaluated based on the conserved region of the apxIVA gene. The results of the exo-probe-based RAA assay were not only determined by real-time fluorescence readout (real-time RAA) but also by a portable blue light instrument for visualization (visual RAA). The results of the nfo-probe-based RAA assay were determined by LFDs (RAA-LFD). These assays could detect all serotypes of A. pleuropneumoniae and had no cross-reaction with other common clinical pathogenic bacteria. The analytical sensitivity of the real-time RAA and RAA-LFD assays was 17.9 copies µL^-1 with a 95% confidence interval, and the analytical sensitivity of the visual RAA assay was 203.5 copies µL^-1 with a 95% confidence interval. The coincidence rate of real-time RAA assay with the qPCR assay was 97.6%, and the coincidence rate of visual RAA and RAA-LFD assays with the qPCR assay was 100% in detecting clinical samples. The RAA assays have high specificity, high sensitivity, and good diagnostic performance, which can be used as reliable diagnostic tools for the rapid detection of A. pleuropneumoniae, especially in on-site diagnostics.

Keywords: Actinobacillus pleuropneumoniae Recombinase-aided amplification quantitative PCR rapid detection

Online: 27 May 2025

Fund:

This study was supported by the University-Industry Collaborative Education Program (220904860093831). We extend our thanks to all the colleagues who participated in this study.

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

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