{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T23:00:10Z","timestamp":1780959610194,"version":"3.54.1"},"reference-count":41,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T00:00:00Z","timestamp":1770854400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"earmarked fund for CARS","award":["CARS36"],"award-info":[{"award-number":["CARS36"]}]},{"name":"innovation fund project of China Animal Health and Epidemiology Center","award":["DW2025004"],"award-info":[{"award-number":["DW2025004"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Brucellosis remains a major global public health concern, with B. melitensis and B. abortus being the primary causative agents in China. This study describes the development of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection and differentiation of B. melitensis and B. abortus. The assay employs TaqMan probes targeting the bcsp31 gene (genus-specific), a transposase gene (B. melitensis-specific) and an autotransporter-associated beta strand repeat-containing protein gene (B. abortus-specific), each labeled with distinct fluorophores (FAM, HEX, ROX). The optimized assay exhibited no cross-reactivity with other pathogens and exhibited significantly higher sensitivity than both qPCR and conventional PCR, with detection limits as low as 2.54\u20133.11 copies\/reaction. Repeatability was excellent, with intra- and inter-assay coefficients of variation below 9%. When validated on a panel of clinical nucleic acid samples, the ddPCR assay showed strong agreement with qPCR (kappa = 0.85), with a sensitivity of 100% (79.42%~100%, 95%CI) and specificity of 95.96% (92.08%~99.84%, 95%CI). These findings establish the multiplex ddPCR as a rapid, sensitive, and highly specific diagnostic platform that improves brucellosis detection accuracy and supports targeted control strategies.<\/jats:p>","DOI":"10.3390\/ani16040566","type":"journal-article","created":{"date-parts":[[2026,2,12]],"date-time":"2026-02-12T12:05:46Z","timestamp":1770897946000},"page":"566","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Development and Evaluation of Multiple Droplet Digital PCR Method for Specific Detection and Differentiation of Brucella melitensis and Brucella abortus"],"prefix":"10.3390","volume":"16","author":[{"given":"Jiwen","family":"Li","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China"},{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jihui","family":"Jin","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuning","family":"Liu","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingjun","family":"Sun","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaqi","family":"Li","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mengkun","family":"Huang","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangxiang","family":"Sun","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4039-9143","authenticated-orcid":false,"given":"Mengda","family":"Liu","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haobo","family":"Zhang","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenlong","family":"Nan","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weixing","family":"Shao","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shufang","family":"Sun","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Yan","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Baoxu","family":"Huang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoxu","family":"Fan","sequence":"additional","affiliation":[{"name":"China Animal Health and Epidemiology Center, Qingdao 266032, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","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. 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