Development and Evaluation of Multiple Droplet Digital PCR Method for Specific Detection and Differentiation of Brucella melitensis and Brucella abortus

Brucellosis remains a major global public health concern, with <i>B. melitensis</i> and <i>B. abortus</i> 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 <i>B. melitensis</i> and <i>B. abortus</i>. The assay employs TaqMan probes targeting the <i>bcsp31</i> gene (genus-specific), a transposase gene (<i>B. melitensis</i>-specific) and an autotransporter-associated beta strand repeat-containing protein gene (<i>B. abortus</i>-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 2.54-3.11 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.