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