Abstract
Brucellosis is a zoonosis caused by Brucella, which poses a great threat to human health and animal husbandry. Pathogen surveillance is an important measure to prevent brucellosis, but the traditional method is time-consuming and not suitable for field applications. In this study, a recombinase polymerase amplification–SYBR Green I (RPAS) assay was developed for the rapid and visualized detection of Brucella in the field by targeting BCSP31 gene, a conserved marker. The method was highly specific without any cross-reactivity with other common bacteria and its detection limit was 2.14 × 104 CFU/mL or g of Brucella at 40 °C for 20 min. It obviates the need for costly instrumentation and exhibits robustness towards background interference in serum, meat, and milk samples. In summary, the RPAS assay is a rapid, visually intuitive, and user-friendly detection that is highly suitable for use in resource-limited settings. Its simplicity and ease of use enable swift on-site detection of Brucella, thereby facilitating timely implementation of preventive measures.
Similar content being viewed by others
Data availability
The authors confirm that the data supporting the findings of this study are available within the article.
References
Atluri VL, Xavier MN, De Jong MF et al (2011) Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol 65:523–541. https://doi.org/10.1146/annurev-micro-090110-102905
Aznar MN, Samartino LE, Humblet M-F, Saegerman C (2014) Bovine brucellosis in Argentina and bordering countries: update. Transbound Emerg Dis 61:121–133. https://doi.org/10.1111/tbed.12018
Baily GG, Krahn JB, Drasar BS, Stoker NG (1992) Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95:271–275
Bruijns BB, Tiggelaar RM, Gardeniers JGE (2016) Fluorescent cyanine dyes for the quantification of low amounts of dsDNA. Anal Biochem 511:74–79. https://doi.org/10.1016/j.ab.2016.07.022
Cao X, Li Z, Liu Z et al (2018) Molecular epidemiological characterization of Brucella isolates from sheep and yaks in northwest China. Transbound Emerg Dis 65:e425–e433. https://doi.org/10.1111/tbed.12777
Chen X, Zhao L, Wang J et al (2022) Rapid visual detection of anisakid nematodes using recombinase polymerase amplification and SYBR Green I. Front Microbiol 13:1026129. https://doi.org/10.3389/fmicb.2022.1026129
Chen Z, Wang Y, Wang Z et al (2013) Improvement and advancement of early diagnosis of human brucellosis in window period. Clin Infect Dis 57:322–323. https://doi.org/10.1093/cid/cit198
Chlebicz A, Śliżewska K (2018) Campylobacteriosis, salmonellosis, yersiniosis, and listeriosis as zoonotic foodborne diseases: A review. Int J Environ Res Public Health 15:863. https://doi.org/10.3390/ijerph15050863
Daher RK, Stewart G, Boissinot M, Bergeron MG (2016) Recombinase polymerase amplification for diagnostic applications. Clin Chem 62:947–958. https://doi.org/10.1373/clinchem.2015.245829
Deqiu S, Donglou X, Jiming Y (2002) Epidemiology and control of brucellosis in China. Vet Microbiol 90:165–182. https://doi.org/10.1016/S0378-1135(02)00252-3
Elsohaby I, Kostoulas P, Elsayed AM et al (2022) Bayesian evaluation of three serological tests for diagnosis of Brucella infections in dromedary camels using latent class models. Prev Vet Med 208:105771. https://doi.org/10.1016/j.prevetmed.2022.105771
Gumaa MM, Cao X, Li Z et al (2019) Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection Mol Cell Probes 47:101434. https://doi.org/10.1016/j.mcp.2019.101434
Khurana SK, Sehrawat A, Tiwari R et al (2021) Bovine brucellosis - a comprehensive review. Vet Q 41:61–88. https://doi.org/10.1080/01652176.2020.1868616
Mayfield JE, Bricker BJ, Godfrey H et al (1988) The cloning, expression, and nucleotide sequence of a gene coding for an immunogenic Brucella abortus protein. Gene 63:1–9. https://doi.org/10.1016/0378-1119(88)90540-9
Pérez-Sancho M, García-Seco T, Arrogante L et al (2013) Development and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of Brucella spp. on clinical samples. Res Vet Sci 95:489–494. https://doi.org/10.1016/j.rvsc.2013.05.002
Riley LW (2020) Extraintestinal foodborne pathogens. Annu Rev Food Sci Technol 11:275–294. https://doi.org/10.1146/annurev-food-032519-051618
Tao J, Liu W, Ding W et al (2020) A multiplex PCR assay with a common primer for the detection of eleven foodborne pathogens. J Food Sci 85:744–754. https://doi.org/10.1111/1750-3841.15033
Whatmore AM, Davison N, Cloeckaert A et al (2014) Brucella papionis sp. nov., isolated from baboons (Papio spp.). Int J Syst Evol Microbiol 64:4120–4128. https://doi.org/10.1099/ijs.0.065482-0
Yagupsky P, Morata P, Colmenero JD (2019) Laboratory diagnosis of human brucellosis. Clin Microbiol Rev 33:e00073-e119. https://doi.org/10.1128/CMR.00073-19
Yamket W, Sathianpitayakul P, Santanirand P, Ratthawongjirakul P (2023) Implementation of helicase-dependent amplification with SYBR Green I for prompt naked-eye detection of bacterial contaminants in platelet products. Sci Rep 13:3238. https://doi.org/10.1038/s41598-023-30410-8
Zhang L, Wu XA, Zhang FL et al (2012) Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies. Mol Biol Rep 39:431–438. https://doi.org/10.1007/s11033-011-0755-9
Zhang S-J, Wang L-L, Lu S-Y et al (2020) A novel, rapid, and simple PMA-qPCR method for detection and counting of viable Brucella organisms. J Vet Res 64:253–261. https://doi.org/10.2478/jvetres-2020-0033
Zheng Y, Hu P, Ren H et al (2021) RPA-SYBR Green I based instrument-free visual detection for pathogenic Yersinia enterocolitica in meat. Anal Biochem 621:114157. https://doi.org/10.1016/j.ab.2021.114157
Funding
This work was supported by grants from the National Key Research and Development Program of China (No. 2021YFC2600202), the Science and Technology Development Project of Jilin Province, China (No. YDZJ202101ZYTS124), the Science and Technology Project of Science and Technology Development Program of Changchun City (No. 21ZGN34), and the Fundamental Research Funds for the Central Universities (No. 45121031B007).
Author information
Authors and Affiliations
Contributions
Jiang Chang participated in methodology, writing and editing. Nan Wang participated in validation. Jun-Peng Zhan participated in writing original draft. Shi-Jun Zhang participated in methodology. De-Ying Zou participated in visualization. Feng Li participated in resources; Ying Zhang participated in investigation. Yan-Song Li participated in supervision. Pan Hu participated in review and editing. Shi-Ying Lu participated in validation. Zeng-Shan Liu participated in formal analysis. Hong-Lin Ren participated in conceptualization, funding acquisition, and project administration. Jiang Chang, Nan Wang, Jun-Peng Zhan, and Shi-Jun Zhang contributed equally in this work.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Chang, J., Wang, N., Zhan, JP. et al. A recombinase polymerase amplification–SYBR Green I assay for the rapid and visual detection of Brucella. Folia Microbiol (2023). https://doi.org/10.1007/s12223-023-01115-2
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s12223-023-01115-2