1. Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort George G. Meade, MD

2. Real-Time PCR Protocols Tick-Borne Pathogens Ehrlichia Species E. chaffeensis, E. ewingii, and Anaplasma phagocytophilum –Hybridization Probes Anaplasma phagocytopilum –Hydrolysis Probes Rickettsia Rickettsia spp. –Hydrolysis Probes

3. Real-Time PCR Protocols continued Tick-Borne Pathogens Borrelia Species B. burgdorferi –Hydrolysis Probes Borrelia spp. –Hydrolysis Probes

4. Conventional PCR Protocols Tick-Borne Pathogens Borrelia Species B. burgdorferi OspA Borrelia spp. FLA Ehrlichia Species E. chaffeensis Anaplasma phagocytophilum

5. Conventional PCR Protocols continued Tick-Borne Pathogens Rickettsia Rickettsia spp. –RFLP to species

6. DETECTION OF ARTHROPOD-BORNE PATHOGENS USING REAL-TIME POLYMERASE CHAIN REACTION TECHNIQUES Hybridization Cleavage Emission FRET Real-time techniques for detection of DNA from tick-borne pathogens Real-time techniques for detection of DNA from tick-borne pathogens Hydrolysis Probes FRET-Fluorescence Resonance Energy Transfer Graphics from the Second Joint Symposium on Food Safety and Nutrition kindly provided by Dr.Guy Van den Eede

17. DETECTION OF ARTHROPOD-BORNE PATHOGENS USING REAL-TIME POLYMERASE CHAIN REACTION TECHNIQUES Real-time techniques for detection of DNA from tick-borne pathogens Real-time techniques for detection of DNA from tick-borne pathogens Hybridization Probes FRET-Fluorescence Resonance Energy Transfer Amplification Hybridization Emission FRET Graphics courtesy of Roche Molecular Biochemicals Inc.

18. Melting Curve Analysis Sequence Confirmation Highly Specific Each product has a specific T M Take advantage of different fluorescent dyes and formats Eliminates inclusion of primer-dimers

19. A. phagocytophila 68.81 ºC E. chaffeensis 62.63 º C E. ewingii 55.15 º C