detection of arthropod-borne pathogens using pcr techniques melissa miller entomologist us army...
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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
E. chaffeensis, E. ewingii, and Anaplasma phagocytophilum
– Hybridization Probes Anaplasma phagocytopilum
– Hydrolysis Probes Rickettsia
Rickettsia spp.– Hydrolysis Probes
Real-Time PCR Protocolscontinued
Tick-Borne Pathogens Borrelia Species
B. burgdorferi– Hydrolysis Probes
Borrelia spp.– Hydrolysis Probes
Conventional PCR Protocols
Tick-Borne Pathogens Borrelia Species
B. burgdorferi OspA Borrelia spp. FLA
Ehrlichia Species E. chaffeensis Anaplasma phagocytophilum
Conventional PCR Protocols continued
Tick-Borne Pathogens Rickettsia
Rickettsia spp.– RFLP to species
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
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
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
AmplificationHybridization
Emission FRET
Graphics courtesy of Roche Molecular Biochemicals Inc.
Melting Curve Analysis
Sequence Confirmation Highly Specific
Each product has a specific TM
Take advantage of different fluorescent dyes and formats
Eliminates inclusion of primer-dimers
A. phagocytophila 68.81 ºC
E. chaffeensis 62.63 º C
E. ewingii55.15 º C