Development and Analytical Evaluation of a Novel Droplet Digital PCR Assay for Molecular Detection of Rickettsia spp. in Dogs and Cats from the Canary Islands

Rickettsia is an obligate intracellular genus of bacteria responsible for rickettsioses, a group of emerging vector-borne diseases. Their diagnosis remains challenging because of non-specific clinical manifestations, serological cross-reactivity, and low bacterial loads in clinical samples. In this study, a novel droplet digital polymerase chain reaction (ddPCR) assay was developed for the molecular detection of Rickettsia spp., and its performance was evaluated using clinical blood samples collected from dogs and cats in the Canary Islands, Spain. Primers and probes targeting the 16S ribosomal RNA (rRNA) were designed and optimized for ddPCR amplification. Analytical performance was assessed by optimising primer and probe concentrations, annealing temperature, specificity, and the limit of detection (LOD). Comparative analysis between quantitative PCR (qPCR) and ddPCR was also performed. The optimized assay employed primer and probe concentrations of 900 nM and 250 nM, respectively, with an annealing temperature of 56 °C. The assay achieved an analytical detection limit of 0.064 ng of clinical sample DNA, although sporadic positive droplets were still detectable at lower concentrations. Strong inverse correlations were observed between qPCR cycle threshold (CT) values and ddPCR absolute quantification for both plasmid DNA (r = −0.997) and clinical sample DNA (r = −0.983). A total of 170 blood samples collected from domestic animals in the Canary Islands, with clinical suspicion of rickettsiosis, were analysed. Six canine samples (3.5%) tested positive for Rickettsia spp. The ddPCR assay developed in this study showed promising analytical performance for the molecular detection of Rickettsia spp. DNA and has the potential to become a valuable molecular tool for veterinary diagnostics and epidemiological investigations within a One Health framework. Further analytical and clinical validation studies are required before its implementation as a routine diagnostic assay.