1. 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

2. 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

3. Mosquito Trapping

4. Human Landing Collections

5. Use of Sentinal Animals

6. Setting Traps During Daylight

7. Setting Traps at Night

8. Reluctant Mosquito Bait

9. Eager to help . . .

10. Early Morning Collecting

11. A Single Trap’s Catch

12. Mosquito Identification
Culex
Anopheles
Aedes
Ochlerotatus
Psorophora
Mansonia
Uranotaenia
Wyomeyia
Coquilletidia

13. 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

14. RNA Extraction Followed by Reverse Transcription
RNA Extraction - Trizol LS
Reverse transcription - formation of cDNA
Cold chain limited to chopped ice

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

16. Equipment Upgrades Refrigerated centrifuge 96-well thermocycler
E-gel™ system

17. Field Gels

18. RAPID Real-Time Cycler
Ruggedized Advanced Pathogen Identification Device

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

20. 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

21. New Technology - MiniOpticon
Benefits: Light weight - 7 kg
48 Samples
Plastic tubes
Conventional PCR
Real-Time PCR

22. Field Use of the MiniOpticon Real-Time Thermocycler

23. 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

24. 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

25. 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

26. From Mosquitoes to PCR Products
Hands-On Demonstration
From Mosquitoes to
PCR Products