Development of Quantitative PCR Assay to Determine Presence of Borrelia burgdorferi within Ixodes scapularis

Kryzsko Commons Ballroom, Winona, Minnesota

Borrelia burgdorferi is the bacterial causative agent of Lyme disease, which is transmitted through the bite of the black-legged tick (Ixodes scapularis). I. scapularis transmits Lyme disease to a wide variety of hosts such as humans, canines, equines, and bovines. Lyme disease is the most prevalent arthropodborne disease in the United States. The focus of this study was to develop a protocol for testing the presence of B.burgdorferi within ticks. DNA was extracted from approximately 6,000 ticks collected between 2005 and 2012 from both Minnesota and Wisconsin. A quantitative PCR (qPCR) was developed using iTaq Universal SYBR green supermix, and the primers of RecA for Borrelia DNA, and ITS2 for the I. scapularis DNA. The ITS2 amplifications served as a control for the viability of the DNA. The RecA amplifications showed if the ticks had B. burgdorferi and were compared to positive and negative controls. The findings to date have shown that the concentration of 1.5µL of RecA per sample, and 0.5µL ITS2 per sample had the best results. The next step will be to continue testing the tick DNA extractions to determine if unknown tick DNA will amplify consistently. Once the protocol is fully developed the prevalence of B. burgdorferi in ticks from 2005-2012 will be analyzed.