Development of a tick-borne pathogen QPCR panel for detection of Anaplasma, Ehrlichia, Rickettsia, and Lyme disease Borrelia in animals.

Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Lyme disease associated Borrelia spp. are the most common tick-borne pathogens reported to infect human beings worldwide and other animals, such as dogs and horses. In the present study, we developed a broad-coverage SYBR Green QPCR panel consisting of four individual assays for the detection and partial differentiation of the aforementioned pathogens. All assays were optimized to the same thermocycling condition and had a detection limit of 10 copies per reaction. The assays remained sensitive when used to test canine and equine blood DNA samples spiked with known amounts of synthetic DNA (gBlock) control template. The assays were specific, as evidenced by lack of cross reaction to non-target gBlock or other pathogens commonly tested in veterinary diagnostic labs. With appropriate Ct cutoff values for positive samples and negative controls and the melting temperature (TM) ranges established in the present study, the QPCR panel is suitable for accurate, convenient and rapid screening and confirmation of tick-borne pathogens in animals.