Development of mRNA-based Hydrolysis Probe TaqMan® real-time Multiplex qPCR assay for Unveiling Active vs. Passive Brucellosis in Caprine Hosts

Background An anthropozoonotic disease, brucellosis is caused by the species of bacteria, Brucella melitensis, affecting small ruminants and other livestock, leading to fiscal losses, and the likelihood of human transmission. Methods This study sought to develop a highly sensitive and specific TaqMan® real-time PCR assay integrating hydrolysis probes for a multiplex assay to differentiate active Brucella infection from passive shedding in goats, targeting specific genes associated with Brucella melitensis . Using discontiguous conserved sequences, primers were designed for B. melitensis genes i.e., omp25, omp31, and IS711 with their compatible fluorescent dyes (Hex, FAM, Texas Red), and quencher molecules (BHQ1, BHQ1, BHQ2), respectively enabling multi-target detection within the same assay. Results The sensitivity of the multiplex assay was evaluated using serial log10 dilutions of Brucella nucleic acid (DNA, showing the presence of bacteria) and (RNA showing the presence of live bacteria), with LOD expressed as absolute copy numbers per reaction. For DNA templates, omp25 was detectable from 8.53×10¹⁰ to 8.53×10⁷ copies, omp31 from 1.32×10¹¹ to 1.32×10⁸, and IS711 from 1.08×10¹¹ to 1.08×10⁷. For RNA (cDNA): omp25 was detected from 1.58×10¹⁰ to 1.58×10⁷ copies, omp31 from 2.45×10¹⁰ to 1.8×10⁶, and IS711 from 2.0×10¹⁰ to 2.0×10⁶. Plasmid-based LODs were 2.6×10⁴, 1.8×10⁵, and 1.6×10⁴ (simplex), and 2.6×10⁴, 1.8×10⁵, and 1.6×10⁵ copies (multiplex). Logistic regression analysis confirmed that omp25 (HEX) had the highest sensitivity with a 95% LOD of approximately 1.95×10⁻⁸ copies. No amplification was observed with Escherichia coli (Gram-negative) or Staphylococcus aureus (Gram-positive), confirming assay specificity. Conclusion However, other abortion-causing microorganisms, such as Listeria monocytogenes , Campylobacter fetus , and Chlamydia abortus , were not tested in this study. Future research should include these pathogens to further validate the specificity of the assay.