Tick-Borne Pathogen Detection Protocol

Blacklegged ticks submitted to the NML through active and passive tick surveillance are routinely tested for DNA or RNA of A. phagocytophilum, Babesia microti, a variety of Borrelia species including B. burgdorferi, and Powassan encephalitis virus by real-time polymerase chain reaction (PCR) as previously described.^{37 (link),38 (link)} Briefly, for the detection of A. phagocytophilum DNA in ticks, we used Qiagen^® DNeasy 96 tissue kits (Qiagen Inc., Mississauga, ON) for DNA extraction as per the manufacturer’s instructions. We eluted DNA in 200 μL of AE buffer and stored at −80°C before use. We used a duplex real-time PCR assay to screen the samples for A. phagocytophilum by targeting the msp2 gene.^{39 (link)}We monitored each round of DNA extractions for cross-contamination by including at least two samples consisting only of nuclease-free water. Synthetic double-stranded DNA controls (Integrated DNA Technologies, Skokie, IL) for Anaplasma were included as positive controls in each PCR run, whereas no-template controls consisting of master mix only served as negative controls. In addition, our positive control DNA for A. phagocytophilum was an equine isolate (MN-93, courtesy of Tim Kurtti, University of Minnesota, MN) that had been propagated in HL-60 promyelocytic cell line (ATCC CCL-240).