USAMRIID Development, Comparison, and Use of Nucleic Acid-Based Diagnostics to Detect Arboviruses in the Field LTC Monica O’Guinn and Dr. John Lee US Army Medical Research Institute of Infectious Disease, Fort Detrick, Frederick, Maryland

Overview Confirmation Detection Sequence Analysis Other Assays Pool Triturate Extract RNA Sequence Analysis TRIzol DNAzol DNA cDNA PCR Ready-To-Go™ Blast Search on GenBank Detection Remaining PCR Product Phylogenetic Tree PCR Clean Up Sequencing

Mosquito Trapping

Human Landing Collections

Use of Sentinal Animals

Setting 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

RAPID Cycler Reactions can be monitored using hybridization probes or double-strand DNA specific dyes, such as SYBR Green

Lightcycler Features STEP #1 Freeze-dried reagents in foil pack STEP #2 Reconstitute with liquid sample or water STEP #3 Load in to R.A.P.I.D STEP #4 Run and read results

New Technology - MiniOpticon Benefits: Light weight - 7 kg 48 Samples Plastic tubes Conventional PCR Real-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 Demonstration From Mosquitoes to PCR Products