FilmArray™ - Nested Multiplex PCR for Multi-pathogen Screening

1. FilmArray™ - Nested Multiplex PCR for Multi-pathogen Screening • Beth Lingenfelter • Regulatory Affairs Manager • Kirk Ririe • CEO, FilmArray Inventor • Mark Poritz, PhD • Senior Scientist • Film Array Program Lead • Todd Ritter • Corporate Development Officer • Marshall McCarty • Director QA/RA

2. Meeting Objectives and Agenda • Objectives • Start a dialogue about FilmArray • Obtain input for product design • Gain insights concerning regulatory issues • Agenda • Introductions and background– Beth Lingenfelter • Principle of operation – Kirk Ririe • Test system evaluation data – Kirk Ririe • Controls – Mark Poritz • Questions – OIVD?

3. Idaho Technology Inc – A PCR company • 1990 – 1995 Rapid Air Thermocyclers • 1996 – ITI launches LightCycler • 1997 – Roche licenses LC from ITI • 1999 – R.A.P.I.D., military version of LC • 2003 – Awarded JBAIDS contract • 2005 – Cleared JBAIDS Anthrax Detection Kit • 2007 – Submitted JBAIDS Plague and Tularemia Detection Kits

4. How does it work? • Nested Multiplex PCR (nmPCR) • High-sensitivity assays for large panels of biological agents. • Chemical Circuits in a Pouch – • Closed system for complex chemistry • Bead Beating to liberate nucleic acids • Magnetic bead nucleic acid extraction • Reverse transcription for RNA targets • Nested Multiplex PCR • High Resolution DNA melting curve analysis • Instrument Automation

5. Pathogen Genome RNA or DNA Nested PCR Outer primers, Reverse transcription, 1st Stage PCR 200bp Dilution Step, Inner primers, 2nd Stage PCR 90bp

6. Primary Multiplex RT-PCR 2nd Stage PCR LCGreen or Taqman 1F 1R 2F 2R 3F 3R 4F 4R 5F 5R 6F 6R Dilute 100 fold Between 1st & 2nd stage reactions Nested Multiplex PCR

7. Robust Multiplex PCR 1 - 5000 x LOD C. burnetti 2 - 5000 x LOD C. burnetti + 0.1x LOD B. anthracis (100 ul of a 100 CFU/ml BA sample)

8. 20 Organism Pool

9. Chemical Circuit-boards Freeze dried reagents for sample preparation and nested multiplex PCR (nmPCR) (A) Reagents delivered into working blisters pneumatically (B) 1st Stage PCR (Blisters F and G) High-density array of 120, 1 ul 2nd stage nmPCR reactions (I)

10. Instrument Automation – Electro-pneumatic Integration • Silicone bladders inflate over blisters to move liquids • Pistons close channels to select flow path • All components integrated in a single core module Air Channels Valve Window - 2nd Stage PCR Pneumatic Bladders

11. Prototype FilmArray™ Instrument • Fully automated • 1 hour to result • System weight 14 lbs + laptop • Five prototype systems in operation

12. The Instrument Generates a Complex Data Set

13. MetaAnalysis Summary

14. Analysis for each organism

15. Analyze the results for each assay

16. System Evaluation Data • Demonstration for US Air Force EOS program, • Lackland AFB, • San Antonio, TX • URI pre-clinical test • Primary Children’s Medical Center (PCMC) • Salt Lake City, UT • IRB approved protocol

17. Lackland AFB Demo – Jul 07 3 Pre-production FilmArray™ Instruments Pooled ATCC samples and frozen de-identified clinical specimens

18. Lackland Results Blinded test demonstrated very reproducible multiplex results across three instruments

19. PCMC Test System Evaluation 100 frozen samples tested at ITI, (collected February-March 2007) 50 Samples tested at PCMC (October – November 2007) Triplicate results are compared to DFA, discordant results are being followed up with sequencing

20. Detection Rates for FilmArray and DFA

21. PCMC Study Results To-date

22. Triple Infection Bocavirus – black PIV2 – blue Human Rhinovirus - green • Bocavirus – black, PIV2 – blue, Human Rhinovirus - green