Process of creating a more efficient real-time quantitative PCR assay to detect Anaplasma phagocytophilum, Babesia microti, Borrelia burgdorferi, and Borrelia miyamotoi that inhabit deer ticks

Ixodes scapularis, the most abundant tick species in New Jersey, is a vector for a host of human diseases, including Lyme disease, Anaplasmosis, Babesiosis, and B. miyamotoi. The prevalence of these diseases continues to steadily increase, and it has become critical to develop molecular methods to detect them. This project focuses on transitioning from a standard PCR and gel electrophoresis methodology to real-time PCR to screen for four common pathogens found in deer ticks – B. burgdorferi, B. miyamotoi, A. phagocytophilum, and B. microti. Primers and probes were created to target genes that were unique to each pathogen. They were tested using standard PCR and gel electrophoresis and were then optimized with real-time PCR using SYBR Green and TaqMan methodologies. The primer specificity was also assessed using tick DNA as a template utilizing a SYBR Green methodology. 158 deer ticks were tested for the presence of pathogens – 11.39% of deer ticks tested positive for B. burgdorferi, 6.33% tested positive for B. microti, 6.96% tested positive for B. miyamotoi, and 10.76% tested positive for A. phagocytophilum. With all of these steps completed, a multiplex PCR assay can be created to screen for all four pathogens in one single test tube.M.S.Includes bibliographical reference