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

## TL;DR

A new digital PCR test was developed to accurately detect and distinguish two types of Brucella bacteria, improving early diagnosis and disease control.

## Contribution

A novel multiplex ddPCR assay for specific detection and differentiation of Brucella melitensis and Brucella abortus with high sensitivity and specificity.

## Key findings

- The ddPCR assay detected as low as 2.54–3.11 copies of Brucella per reaction with high reproducibility.
- The assay showed superior sensitivity compared to qPCR, detecting 17 positives versus 13 in clinical samples.
- The method achieved 100% sensitivity and 95.96% specificity in clinical validation.

## Abstract

Brucellosis is a widespread and highly contagious zoonotic disease with serious implications for both animal and human health. In China, Brucella melitensis and Brucella abortus are the predominant species, accounting for 80–90% of human brucellosis cases. This study presents the development and validation of novel multiplex droplet digital PCR (ddPCR) assay designed for the highly sensitive and specific detection and differentiation of B. melitensis and B. abortus. The assay achieved exceptional detection limits of 3.11 copies/reaction (95% CI: 2.68–3.61) for B. melitensis and 2.54 copies/reaction (95%CI: 2.32–2.79) for B. abortus, with strong linearity (R2 > 0.99) and high reproducibility (CV < 9%). In a comparative evaluation using a panel of clinical nucleic acid samples, ddPCR detected 17 positives versus 13 by qPCR, demonstrating superior sensitivity, particularly in samples with low bacterial load. This robust and precise multiplex ddPCR assay provides a reliable tool for early diagnosis and epidemiological surveillance, enabling improved brucellosis control.

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.