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LTC Monica O’Guinnand
Dr. John LeeUS Army Medical Research Institute of Infectious Disease,
Fort Detrick, Frederick, Maryland
USAMRIIDUSAMRIID
Development, Comparison, and Use of Nucleic Acid-Based
Diagnostics to Detect Arboviruses in the Field
Development, Comparison, and Use of Nucleic Acid-Based
Diagnostics to Detect Arboviruses in the Field
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OverviewOverviewExtractExtract RNARNA
cDNAcDNA
PCRPCR
DetectionDetection
Pool
Triturate
Pool
Triturate TRIzolTRIzol
DNADNADNAzolDNAzol
Ready-To-Go™Ready-To-Go™
Other AssaysOther Assays
PCR Clean Up
Sequencing
Sequence Analysis
Blast Search on GenBank
Phylogenetic TreeRemaining PCR Product
Confirmation
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Mosquito Trapping
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Human Landing Collections
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Use of Sentinal Animals
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Setting Traps During DaylightSetting Traps During Daylight
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Setting Traps at Night
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Reluctant Mosquito Bait
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Eager to help . . .
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Early Morning Collecting
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A Single Trap’s Catch
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Mosquito Identification
• Culex • Anopheles• Aedes• Ochlerotatus• Psorophora• Mansonia• Uranotaenia• Wyomeyia• Coquilletidia
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Mosquito Preparation
• Pool mosquitoes by species
• Triturate mosquitoes in PBS/media
• Add 250 ul of mosquito suspension to 750 ul of TRIzol® LS
• Use of BBs to triturate-decreases contamination
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RNA Extraction Followed by Reverse Transcription
• RNA Extraction - Trizol LS
• Reverse transcription - formation of cDNA
• Cold chain limited to chopped ice
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Equipment• Thermocycler• Transilluminator• Electrophoresis unit• Microscope• Centrifuge• Camera • RT reagents• PCR reagents• Ice chest/cold block• Pipettors
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Equipment Upgrades
• Refrigerated centrifuge
• 96-well thermocycler
• E-gel™ system
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Field Gels
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RAPID Real-Time Cycler
Ruggedized Advanced Pathogen Identification Device
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Reactions can be monitored using hybridization probes or double-strand DNA specific dyes, such as SYBR Green
Reactions can be monitored using hybridization probes or double-strand DNA specific dyes, such as SYBR Green
RAPID CyclerRAPID Cycler
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Lightcycler FeaturesLightcycler FeaturesSTEP #1Freeze-dried reagents in foil packSTEP #1Freeze-dried reagents in foil pack
STEP #2Reconstitute with liquid sample or waterSTEP #2Reconstitute with liquid sample or water
STEP #3Load in to R.A.P.I.DSTEP #3Load in to R.A.P.I.D
STEP #4Run and read resultsSTEP #4Run and read results
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New Technology - MiniOpticon
Conventional PCRConventional PCR
Benefits: Light weight - 7 kg48 SamplesPlastic tubes
Benefits: Light weight - 7 kg48 SamplesPlastic tubes
Real-Time PCRReal-Time PCR
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Field Use of the MiniOpticon Real-Time Thermocycler
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Conventional PCR• Pros
• Well established technology• Product amenable to direct sequencing• Can visualize band intensity and size• Literature contains multiple references • Present in the Army inventory• 96-well block
• Cons• Requires gels• Longer time
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Real-Time PCR - RAPID
• Advantages• Rapid heating of sample - shorter run times• Present in the Army inventory
• Disadvantages• Glass capillaries and 32-reactions per run• Heavy - difficult as checked baggage• Expensive - fluorescently labeled probes• No automatic memory after power failure
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Real-Time PCR - MiniOpticon
• Advantages• Plastic tubes and • Lightweight instrument• Dual use for real-time and conventional
• Disadvantages• Overheats easily • Sun glare sensitive• No automatic memory
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From Mosquitoes to
PCR Products
Hands-On DemonstrationHands-On Demonstration