![Page 1: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/1.jpg)
Detection of Arthropod-Borne Pathogens Using PCR Techniques
Melissa Miller Entomologist
US Army Center for Health Promotion & Preventive Medicine,
Fort George G. Meade, MD
![Page 2: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/2.jpg)
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
![Page 3: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/3.jpg)
Real-Time PCR Protocolscontinued
Tick-Borne Pathogens Borrelia Species
B. burgdorferi– Hydrolysis Probes
Borrelia spp.– Hydrolysis Probes
![Page 4: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/4.jpg)
Conventional PCR Protocols
Tick-Borne Pathogens Borrelia Species
B. burgdorferi OspA Borrelia spp. FLA
Ehrlichia Species E. chaffeensis Anaplasma phagocytophilum
![Page 5: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/5.jpg)
Conventional PCR Protocols continued
Tick-Borne Pathogens Rickettsia
Rickettsia spp.– RFLP to species
![Page 6: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/6.jpg)
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
![Page 7: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/7.jpg)
![Page 8: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/8.jpg)
![Page 9: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/9.jpg)
![Page 10: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/10.jpg)
![Page 11: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/11.jpg)
![Page 12: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/12.jpg)
![Page 13: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/13.jpg)
![Page 14: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/14.jpg)
![Page 15: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/15.jpg)
![Page 16: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/16.jpg)
![Page 17: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/17.jpg)
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.
![Page 18: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/18.jpg)
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
![Page 19: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/19.jpg)
A. phagocytophila 68.81 ºC
E. chaffeensis 62.63 º C
E. ewingii55.15 º C
![Page 20: Detection of Arthropod-Borne Pathogens Using PCR Techniques Melissa Miller Entomologist US Army Center for Health Promotion & Preventive Medicine, Fort](https://reader036.vdocuments.net/reader036/viewer/2022062314/56649ebd5503460f94bc6ff8/html5/thumbnails/20.jpg)