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

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

Mosquito Trapping

Human Landing Collections

Use of Sentinal Animals

Setting Traps During DaylightSetting Traps During Daylight

Setting Traps at Night

Reluctant Mosquito Bait

Eager to help . . .

Early Morning Collecting

A Single Trap’s Catch

Mosquito Identification

• Culex • Anopheles• Aedes• Ochlerotatus• Psorophora• Mansonia• Uranotaenia• Wyomeyia• Coquilletidia

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

RNA Extraction Followed by Reverse Transcription

• RNA Extraction - Trizol LS

• Reverse transcription - formation of cDNA

• Cold chain limited to chopped ice

Equipment• Thermocycler• Transilluminator• Electrophoresis unit• Microscope• Centrifuge• Camera • RT reagents• PCR reagents• Ice chest/cold block• Pipettors

Equipment Upgrades

• Refrigerated centrifuge

• 96-well thermocycler

• E-gel™ system

Field Gels

RAPID Real-Time Cycler

Ruggedized Advanced Pathogen Identification Device

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

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

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

Field Use of the MiniOpticon Real-Time Thermocycler

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

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

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

From Mosquitoes to

PCR Products

Hands-On DemonstrationHands-On Demonstration