Detection of Arthropod-Borne Pathogens Using PCR Techniques

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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

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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

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Real-Time PCR Protocols continued

• Tick-Borne Pathogens
• Borrelia Species
• B. burgdorferi
• Hydrolysis Probes
• Borrelia spp.
• Hydrolysis Probes

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Conventional PCR Protocols

• Tick-Borne Pathogens
• Borrelia Species
• B. burgdorferi OspA
• Borrelia spp. FLA
• Ehrlichia Species
• E. chaffeensis
• Anaplasma phagocytophilum

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Conventional PCR Protocols continued

• Tick-Borne Pathogens
• Rickettsia
• Rickettsia spp.
• RFLP to species

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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
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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.
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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

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