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Detection of Arthropod-Borne Pathogens Using PCR Techniques

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

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Detection of Arthropod-Borne Pathogens Using PCR Techniques

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  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 Protocolscontinued • 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 • Real-time techniques for detection of DNA from tick-borne pathogens • Hydrolysis Probes • FRET-Fluorescence Resonance Energy Transfer Emission FRET Hybridization Cleavage Graphics from the Second Joint Symposium on Food Safety and Nutrition kindly provided by Dr.Guy Van den Eede

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

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

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

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