{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T19:28:27Z","timestamp":1792092507546,"version":"4.4.0"},"reference-count":30,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T00:00:00Z","timestamp":1759276800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:p>Brucellosis is a zoonosis that occurs worldwide, and vaccination is the main strategy for controlling it. In China, the <jats:italic>Brucella abortus<\/jats:italic> A19-\u0394VirB12 strain is utilized in main vaccines. However, a high-sensitivity nucleic acid detection method to effectively differentiate <jats:italic>Brucella<\/jats:italic> infections from immunization with the A19-\u0394VirB12 strain is lacking. Therefore, in this study, a duplex droplet digital PCR (ddPCR) assay was established using primers and probes targeting the <jats:italic>VirB8<\/jats:italic> gene and the deleted <jats:italic>VirB12<\/jats:italic> gene in the A19-\u0394VirB12 strain. The specificity of the method was tested using genomic DNA of <jats:italic>Mycobacterium bovis<\/jats:italic>, <jats:italic>Escherichia coli<\/jats:italic> (O:157), <jats:italic>Salmonella<\/jats:italic> spp., <jats:italic>Streptococcus<\/jats:italic> spp., and A19-\u0394VirB12 <jats:italic>Brucella<\/jats:italic>. Only A19-\u0394VirB12 amplified <jats:italic>VirB8<\/jats:italic> gene. The detection limits of the method for <jats:italic>VirB8<\/jats:italic> and <jats:italic>VirB12<\/jats:italic> were 2.13\u202f\u00d7\u202f10<jats:sup>0<\/jats:sup> and 2.26\u202f\u00d7\u202f10<jats:sup>0<\/jats:sup> copies\/\u03bcL, respectively. In the detection of DNA in epidemic-related samples, the positive rate of ddPCR was much higher than that in the samples analyzed using the commercial fluorescence quantitative reagent kits. Meanwhile, the ddPCR of the A19-\u0394VirB12 <jats:italic>Brucella<\/jats:italic> vaccine strain was identified in the clinical samples. In summary, the ddPCR method with high sensitivity and specificity was established, which will support the future identification of A19-\u0394VirB12 <jats:italic>Brucella<\/jats:italic> vaccine strains in immunized and wild-type <jats:italic>Brucella<\/jats:italic>.<\/jats:p>","DOI":"10.3389\/fmicb.2025.1684156","type":"journal-article","created":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T05:49:21Z","timestamp":1759297761000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Establishment of a dual droplet digital PCR method for detecting the Brucella abortus A19-\u0394VirB12 strains"],"prefix":"10.3389","volume":"16","author":[{"given":"Ruixue","family":"Xue","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zunfeng","family":"Chu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linlin","family":"Xing","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zixin","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenduo","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yingli","family":"Shang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangkun","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongmei","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuyu","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanling","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Miao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinglin","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingjun","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zouran","family":"Lan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yue","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,10,1]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"109007","DOI":"10.1016\/j.vetmic.2021.109007","article-title":"Characteristics of Brucella abortus vaccine strain A19 reveals its potential mechanism of attenuated virulence","volume":"254","author":"Cheng","year":"2021","journal-title":"Vet. Microbiol."},{"key":"ref2","first-page":"1","volume-title":"Brucellosis in humans and animals","author":"Corbel","year":"2006"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"39","DOI":"10.1016\/j.ijfoodmicro.2018.12.009","article-title":"Human brucellosis caused by raw dairy products: a review on the occurrence, major risk factors and prevention","volume":"292","author":"Dadar","year":"2019","journal-title":"Int. J. Food Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"e1929","DOI":"10.1371\/journal.pntd.0001929","article-title":"Clinical manifestations of human brucellosis: a systematic review and meta-analysis","volume":"6","author":"Dean","year":"2012","journal-title":"PLoS Negl. Trop. Dis."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"e0010468","DOI":"10.1371\/journal.pntd.0010468","article-title":"Methodological choices in brucellosis burden of disease assessments: a systematic review","volume":"16","author":"Di Bari","year":"2022","journal-title":"PLoS Negl. Trop. Dis."},{"key":"ref6","doi-asserted-by":"publisher","first-page":"54","DOI":"10.5312\/wjo.v10.i2.54","article-title":"Osteoarticular manifestations of human brucellosis: a review","volume":"10","author":"Esmaeilnejad-Ganji","year":"2019","journal-title":"World J. Orthop."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1016\/S1473-309(07)70286-4","article-title":"Human brucellosis","volume":"7","author":"Franco","year":"2007","journal-title":"Lancet Infect. Dis."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.bdq.2015.12.003","article-title":"Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms","volume":"7","author":"Gerdes","year":"2016","journal-title":"Biomol. Detect. Quantif."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"349","DOI":"10.18869\/acadpub.ibj.21.6.349","article-title":"A review of brucellosis in Iran: epidemiology, risk factors, diagnosis, control, and prevention","volume":"21","author":"Golshani","year":"2017","journal-title":"Iran. Biomed. J."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1186\/s12917-022-03229-0","article-title":"Combined immunization with inactivated vaccine reduces the dose of live B. abortus A19 vaccine","volume":"18","author":"He","year":"2022","journal-title":"BMC Vet. Res."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"149","DOI":"10.21608\/EJVS.2024.259043.1751","article-title":"Comparable study of immunological, bacteriological, and molecular techniques for detecting brucellosis in milk of reproductively problematic cows","volume":"56","author":"Helmy","year":"2025","journal-title":"J. Vet. Sci."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1016\/j.jviromet.2012.08.019","article-title":"Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR","volume":"186","author":"Henrich","year":"2012","journal-title":"J. Virol. Methods"},{"key":"ref13","doi-asserted-by":"publisher","first-page":"8604","DOI":"10.1021\/ac202028g","article-title":"High-throughput droplet digital PCR system for absolute quantitation of DNA copy number","volume":"83","author":"Hindson","year":"2011","journal-title":"Anal. Chem."},{"key":"ref14","doi-asserted-by":"publisher","first-page":"1789","DOI":"10.3201\/eid2909.230052","article-title":"Global estimate of human brucellosis incidence","volume":"29","author":"Laine","year":"2023","journal-title":"Emerg. Infect. Dis."},{"key":"ref15","doi-asserted-by":"publisher","first-page":"113875","DOI":"10.1016\/j.jviromet.2020.113875","article-title":"Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus","volume":"280","author":"Lin","year":"2020","journal-title":"J. Virol. Methods"},{"key":"ref16","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1007\/978-1-4939-8837-2_14","article-title":"Quantitative analysis with droplet digital PCR","volume":"1875","author":"Mehle","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"4310","DOI":"10.1038\/s41598-021-83723-x","article-title":"Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients","volume":"22","author":"Paolo","year":"2021","journal-title":"Sci. Rep."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"91","DOI":"10.1016\/S1473-3099(06)70382-6","article-title":"The new global map of human brucellosis","volume":"6","author":"Pappas","year":"2006","journal-title":"Lancet Infect. Dis."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"4945","DOI":"10.1128\/IAI.00584-06","article-title":"Swapping of periplasmic domains between Brucella suis VirB8 and a pSB102 VirB8 homologue allows heterologous complementation","volume":"74","author":"Patey","year":"2006","journal-title":"Infect. Immun."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"379","DOI":"10.1111\/j.1574-6976.2010.00211.x","article-title":"Towards a Brucella vaccine for humans","volume":"34","author":"Perkins","year":"2010","journal-title":"FEMS Microbiol. Rev."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"1271","DOI":"10.3390\/s18041271","article-title":"dPCR: a technology review","volume":"18","author":"Quan","year":"2018","journal-title":"Sensors"},{"key":"ref22","doi-asserted-by":"publisher","first-page":"1075","DOI":"10.1128\/IAI.71.3.1075-1082.2003","article-title":"Production of the type IV secretion system differs among Brucella species as revealed with VirB5- and VirB8-specific antisera","volume":"71","author":"Rouot","year":"2003","journal-title":"Infect. Immun."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"931","DOI":"10.3390\/diagnostics14090931","article-title":"Present and future applications of digital PCR in infectious diseases diagnosis","volume":"14","author":"Sancha Dominguez","year":"2024","journal-title":"Diagnostics"},{"key":"ref24","doi-asserted-by":"publisher","first-page":"392","DOI":"10.1016\/j.vetmic.2009.06.021","article-title":"Brucellosis: a re-emerging zoonosis","volume":"140","author":"Seleem","year":"2010","journal-title":"Vet. Microbiol."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"843","DOI":"10.1590\/1414-431X20154243","article-title":"A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain","volume":"48","author":"Weiss","year":"2015","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"7583","DOI":"10.1007\/s00216-016-9861-9","article-title":"Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR","volume":"408","author":"Witte","year":"2016","journal-title":"Anal. Bioanal. Chem."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"679560","DOI":"10.3389\/fimmu.2021.679560","article-title":"Evaluation and differential diagnosis of a genetic marked Brucella vaccine A19\u0394virB12 for cattle","volume":"12","author":"Yang","year":"2021","journal-title":"Front. Immunol."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"60","DOI":"10.1007\/s11515-012-1196-0","article-title":"Progress in Brucella vaccine development","volume":"8","author":"Yang","year":"2013","journal-title":"Front. Biol."},{"key":"ref29","first-page":"1213","article-title":"Construction of Brucella abortus A19-delta VirB12 mutant and evaluation of its protective efficacy against 2308 strain challenge in BALB\/c mice","volume":"53","author":"Yi","year":"2013","journal-title":"Wei Sheng Wu Xue Bao"},{"key":"ref30","doi-asserted-by":"publisher","first-page":"821","DOI":"10.1093\/clinids\/5.5.821","article-title":"Human brucellosis","volume":"5","author":"Young","year":"1983","journal-title":"Rev. Infect. 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