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ORIGINAL RESEARCH article

Front. Cell. Infect. Microbiol.
Sec. Clinical Microbiology
Volume 14 - 2024 | doi: 10.3389/fcimb.2024.1493492
This article is part of the Research Topic Pathogenic Mechanisms and New Technology-Based Diagnostics for Bacterial Infections View all articles

Establishment of a rapid method for the detection of Brucella canis based on recombinasemediated thermostable nucleic acid amplification technology

Provisionally accepted
Shao-Zheng Song Shao-Zheng Song 1*Zi-Yuan Li Zi-Yuan Li 1*Yuanyuan Liu Yuanyuan Liu 1Ying-Chao Wu Ying-Chao Wu 2Kang-Ying Yu Kang-Ying Yu 1*
  • 1 Taihu University of Wuxi, Wuxi, Jiangsu, China
  • 2 Jiangyin Lingfeng pet Hospital, Wuxi, China

The final, formatted version of the article will be published soon.

    Objective: To establish a rapid detection method for canine brucellosis using recombinase-aided amplification (RAA) technology.Methods :The outer membrane protein 25 gene fragment (Omp25) of Brucella 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 Brucella canis, Brucella suis, Brucella melitensis, Brucella abortus, Staphylococcus aureus, pathogenic Escherichia coli, Salmonella enteritidis, Shigella spp., Proteus mirabilis, and Listeria monocytogenes. Reproducibility was evaluated using plasmid standards from the same and different batches.Results: The optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39℃ for 15 minutes. The detection sensitivity was 1 copy/μL. No cross-reaction was observed with other Brucella 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 Brucella 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 brucellosis and the precise diagnosis of other pet diseases, making it suitable for promotion and application.

    Keywords: recombinase, Thermostatic, Nucleic acid amplification, Rapid detection, Brucella canis

    Received: 09 Sep 2024; Accepted: 25 Nov 2024.

    Copyright: © 2024 Song, Li, Liu, Wu and Yu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Shao-Zheng Song, Taihu University of Wuxi, Wuxi, Jiangsu, China
    Zi-Yuan Li, Taihu University of Wuxi, Wuxi, Jiangsu, China
    Kang-Ying Yu, Taihu University of Wuxi, Wuxi, Jiangsu, China

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