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 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 2.54–3.11 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.