Validation of Droplet Digital PCR (ddPCR) for the Detection and Absolute Quantification of Borrelia DNA in Ixodes Ticks

In this research, QX200 Droplet Digital PCR (ddPCRTM) system protocols for the detection of bacterial (Borrelia burgdorferi and Borrelia miyamotoi) DNA were developed and tested. Existing Ixodes scapularis samples collected from Cape Cod, Massachusetts and previously determined to be 60% positive for B. burgdorferi were utilized to investigate absolute bacterial genome carriage per tick using the ddPCR assays optimized here. The ddPCR technology proved to be a reliable means for detection and absolute quantification of control bacterial DNA with sensitivity as low as 10 spirochetes per μl input DNA. Application of ddPCR revealed an average B. burgdorferi carriage level of 27,239 copies in infected ticks (range: 231- 118,407 copies), 2,197 copies in infected nymphs (range: 231- 4,983 copies), and 45,620 copies in infected adults (range: 5,647- 118,407 copies). This is the first known and validated application of ddPCR for the detection of Borrelia DNA in Ixodes ticks.