USAMRIID Development, Comparison, and Use of Nucleic Acid-Based Diagnostics to Detect Arboviruses in the Field LTC Monica O’Guinn and Dr.
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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 Pool Triturate Other Assays Extract TRIzol RNA Confirmation Sequence Analysis DNAzol DNA cDNA PCR Ready-To-Go™ Detection Remaining PCR Product PCR Clean Up Sequencing Blast Search on GenBank Phylogenetic Tree 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 trituratedecreases 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 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 Lightcycler Features 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 Hands-On Demonstration From Mosquitoes to PCR Products