Design, validation and evaluation of a triplex real time qualitative PCR assay targeting zoonotic Capnocytophaga species in EDTA whole blood and plasma simulated clinical specimens

Found in the oral microbiome of cats and dogs, bacteria from the genus Capnocytophaga occasionally infect humans and can cause serious disease or death if not identified and treated promptly. Described here is a real-time triplex qualitative PCR assay designed using gene targets (rplV and secY) identified as diagnostically relevant by analysis of genomes from the Capnocytophaga species. The assay was evaluated on simulated EDTA whole blood samples/plasma and is effective at detecting down to 10 copies per microliter (10000 copies per mL, 4 log/mL) for whole blood samples (rplV /secY), and 1 copy per microliter (1000 copies per mL, 3 log mL) (rplV) and 0.5 copy per microliter (500 copies per mL, 2.69 log mL) (secY) for plasma samples. Analytical sensitivity, specificity, and reproducibility were established in plasmid-spiked simulated EDTA whole blood and plasma specimens, and clinical performance in Capnocytophaga-positive patient samples remains to be determined.