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In addition to traditional serological tests and bacterial culture, the emergence of molecular tests, particularly quantitative PCR (qPCR), has gained attention for their rapid turnaround time, as well as their high sensitivity and specificity. However, validating existing assays using a sufficiently large and diverse data set remains challenging, and there is a pressing need for a reliable multiplex assay capable of simultaneously identifying\n B. abortus<\/jats:italic>\n ,\n B. melitensis<\/jats:italic>\n , and\n B. suis<\/jats:italic>\n . This study reports the development and evaluation of a highly sensitive and specific multiplex qPCR assay for detecting three\n Brucella<\/jats:italic>\n species, alongside a novel genus-specific\n Brucella<\/jats:italic>\n qPCR assay, both designed through large-scale comparative analysis of whole-genome sequencing (WGS) data.\n In silico<\/jats:italic>\n validation of these primer-probe sets demonstrated sensitivities and specificities ranging from 99.8% to 100% using a large WGS data set, indicating comparable or improved\n in silico<\/jats:italic>\n performance relative to currently published qPCR assays. Subsequently, the qPCR assays were evaluated using a comprehensive genomic DNA panel comprising 235 classical\n Brucella<\/jats:italic>\n strains and 192 non-classical\n Brucella<\/jats:italic>\n or non-\n Brucella<\/jats:italic>\n samples, achieving 100% sensitivity and specificity with excellent low-level detection capability. Furthermore, these assays successfully detected\n Brucella<\/jats:italic>\n DNA in tissue samples, demonstrating exceptional sensitivity and diagnostic accuracy through an advanced bacterial DNA enrichment process. These qPCR assays represent a valuable tool for rapid, accurate diagnosis of\n Brucella<\/jats:italic>\n species in clinical specimens and bacterial cultures.\n <\/jats:p>\n \n IMPORTANCE<\/jats:title>\n \n Brucellosis is a disease affecting animals and humans, causing major health and economic problems worldwide. It is mainly caused by\n Brucella abortus<\/jats:italic>\n ,\n Brucella melitensis<\/jats:italic>\n , and\n Brucella suis<\/jats:italic>\n . Quick and accurate identification of these bacteria is critical to control the spread of infection and protect public health. Current laboratory tests have limited ability to detect the previously mentioned\n Brucella<\/jats:italic>\n species simultaneously and often lack validation across large and diverse sample sets, indicating a need for enhanced diagnostic tools. This study presents the development and evaluation of two rapid, accurate molecular tests that can detect the\n Brucella<\/jats:italic>\n genus or precisely identify these three critical species simultaneously, thereby saving time and resources. With their excellent performance on large data sets across both computer-based and laboratory environments, these tests offer hope for improved diagnosis in both clinical and veterinary applications, thereby supporting public health and animal disease control efforts.\n <\/jats:p>\n <\/jats:sec>\n <\/jats:sec>","DOI":"10.1128\/spectrum.02974-25","type":"journal-article","created":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T13:01:11Z","timestamp":1774530071000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Two novel real-time PCR assays for\n Brucella<\/i>\n detection: a species-specific multiplex and a genus-level singleplex developed via large-scale whole genome sequencing data 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