LTC Monica OGuinn

1
Development, Comparison, and Use of Nucleic
Acid-Based Diagnostics to Detect Arboviruses in
the Field

• LTC Monica OGuinn
• and
• Dr. John Lee
• US Army Medical Research Institute of Infectious
  Disease, Fort Detrick, Frederick, Maryland

2
Overview
Confirmation
Other Assays
Pool Triturate
RNA
Extract
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 Traps 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 1Freeze-dried reagents in foil pack
STEP 2Reconstitute with liquid sample or water
STEP 3Load in to R.A.P.I.D
STEP 4Run 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
Hands-On Demonstration

• From Mosquitoes to
• PCR Products