Detection and quantification of <i>Brucella abortus</i> DNA in water buffaloes ( <i>bubalus bubalis</i> ) using droplet digital polymerase chain reaction

Brucellosis represents a major public health concern worldwide. Human transmission is mainly due to the consumption of unpasteurized milk and dairy products of infected animals. The gold standard for the diagnosis of <i>Brucella</i> spp in ruminants is the bacterial isolation, but it is time-consuming. Polymerase Chain Reaction (PCR) is a quicker and more sensitive technique than bacterial culture. Droplet digital PCR (ddPCR) is a novel molecular assay showing high sensitivity in samples with low amount of DNA and lower susceptibility to amplification inhibitors. Present study aimed to develop a ddPCR protocol for the detection of <i>Brucella abortus</i> in buffalo tissue samples. The protocol was validated using proficiency test samples for <i>Brucella</i> spp by real time qPCR. Furthermore, 599 tissue samples were examined. Among reference materials, qPCR and ddPCR demonstrated same performance and were able to detect up to 225 CFU/mL. Among field samples, ddPCR showed higher sensitivity (100%), specificity and accuracy of 93.4% and 94.15%, respectively. ddPCR could be considered a promising technique to detect <i>B. abortus</i> in veterinary specimens, frequently characterized by low amount of bacteria, high diversity in matrices and species and poor storage conditions.