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

Meeting Objectives and Agenda 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?

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

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

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

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

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)

20 Organism Pool

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)

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

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

The Instrument Generates a Complex Data Set

MetaAnalysis Summary

Analysis for each organism

Analyze the results for each assay

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

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

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

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

Detection Rates for FilmArray and DFA

PCMC Study Results To-date

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