Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology
ObjectiveTo establish a rapid detection method for canine brucellosis using recombinase-aided amplification (RAA) technology.MethodsThe outer membrane protein 25 gene fragment (Omp25) of Brucella canis was targeted. Primers and fluorescent probes were designed and synthesized, and recombinant plasmi...
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Frontiers Media S.A.
2025-01-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1493492/full |
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| author | Shao-Zheng Song Zi-Yuan Li Yuan-Yuan Liu Ying-Chao Wu Kang-Ying Yu Zhengyi He |
| author_facet | Shao-Zheng Song Zi-Yuan Li Yuan-Yuan Liu Ying-Chao Wu Kang-Ying Yu Zhengyi He |
| author_sort | Shao-Zheng Song |
| collection | DOAJ |
| description | ObjectiveTo establish a rapid detection method for canine brucellosis using recombinase-aided amplification (RAA) technology.MethodsThe 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.ResultsThe optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39°C 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).ConclusionThe 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. |
| format | Article |
| id | doaj-art-90251cc2d1e1443cb33fd4d85c26e933 |
| institution | Kabale University |
| issn | 2235-2988 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-90251cc2d1e1443cb33fd4d85c26e9332025-01-03T06:46:54ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-01-011410.3389/fcimb.2024.14934921493492Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technologyShao-Zheng Song0Zi-Yuan Li1Yuan-Yuan Liu2Ying-Chao Wu3Kang-Ying Yu4Zhengyi He5School of Health and Nursing/Department of Basic, Wuxi Taihu University, Wuxi, Jiangsu, ChinaSchool of Health and Nursing/Department of Nursing, Wuxi Taihu University, Wuxi, Jiangsu, ChinaSchool of Health and Nursing/Department of Nursing, Wuxi Taihu University, Wuxi, Jiangsu, ChinaDepartment of Internal Medicine, Jiangyin Lingfeng Pet Hospital, Wuxi, Jiangsu, ChinaSchool of Health and Nursing/Department of Basic, Wuxi Taihu University, Wuxi, Jiangsu, ChinaThe First Affiliated Hospital of Gannan Medical University, Ganzhou, Jiangxi, ChinaObjectiveTo establish a rapid detection method for canine brucellosis using recombinase-aided amplification (RAA) technology.MethodsThe 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.ResultsThe optimized RAA system used primers bOmp25-F2/bOmp25-R2 and probe bOmp25-P, with a constant reaction temperature of 39°C 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).ConclusionThe 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.https://www.frontiersin.org/articles/10.3389/fcimb.2024.1493492/fullrecombinasethermostaticnucleic acid amplificationrapid detectionBrucella canis |
| spellingShingle | Shao-Zheng Song Zi-Yuan Li Yuan-Yuan Liu Ying-Chao Wu Kang-Ying Yu Zhengyi He Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology Frontiers in Cellular and Infection Microbiology recombinase thermostatic nucleic acid amplification rapid detection Brucella canis |
| title | Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology |
| title_full | Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology |
| title_fullStr | Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology |
| title_full_unstemmed | Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology |
| title_short | Establishment of a rapid method for the detection of Brucella canis based on recombinase-mediated thermostable nucleic acid amplification technology |
| title_sort | establishment of a rapid method for the detection of brucella canis based on recombinase mediated thermostable nucleic acid amplification technology |
| topic | recombinase thermostatic nucleic acid amplification rapid detection Brucella canis |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2024.1493492/full |
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