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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.

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filmarray nested multiplex pcr for multi pathogen screening
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
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?
idaho technology inc a pcr company
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
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

Nested PCR

Outer primers,

Reverse transcription,

1st Stage PCR

200bp

Dilution Step,

Inner primers, 2nd Stage PCR

90bp

nested multiplex pcr

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
robust multiplex 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)

chemical circuit boards
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
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
Prototype FilmArray™ Instrument
  • Fully automated
  • 1 hour to result
  • System weight 14 lbs + laptop
  • Five prototype systems in operation
system evaluation data
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
Lackland AFB Demo – Jul 07

3 Pre-production FilmArray™ Instruments

Pooled ATCC samples and frozen de-identified clinical specimens

lackland results
Lackland Results

Blinded test demonstrated very reproducible multiplex results across three instruments

pcmc test system evaluation
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

triple infection
Triple Infection

Bocavirus – black

PIV2 – blue

Human Rhinovirus - green

  • Bocavirus – black, PIV2 – blue, Human Rhinovirus - green