Transcript Document

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
1
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?
2
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
3
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
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Nested PCR
Pathogen Genome
RNA or DNA
Outer primers,
Reverse transcription,
1st Stage PCR
200bp
Dilution Step,
Inner primers, 2nd Stage PCR
90bp
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Nested Multiplex PCR
Primary Multiplex RT-PCR
1F
2nd Stage PCR
1R
2F
LCGreen or Taqman
2R
3F
3R
4F
4R
5F
5R
6F
6R
Dilute 100 fold
Between 1st & 2nd stage reactions
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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)
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20 Organism Pool
20 Organism Mixture Results
Organism
Subtype
Adenovirus
Phylo group1
Phylo group2
Phylo group3
Phylo group4
Bocavirus
Coronavirus
SARS
Coronavirus
229E
Coronavirus
NL63
Coronavirus
OC43
Coronavirus
HKU1
Pan
Influenza A
H3N2
H5N1
Pan
Influenza B
Human
Metapneumov
Phylo group1
Human
Phylo group2
Rhinovirus
Phylo group3
Phylo group4
Parainfluenza 1
2
virus
3
4
RSV
pertussis
Bordatella
pneumoniae
Clymidia
Mycoplasma pneumoniae
Gene
Hexon
Hexon
Hexon
Hexon
NP
NS
NG
Pol
NG
Pol
NG
Pol
NG
Pol
NG
Pol
Matrix
HA
NA
HA
NA
HA
NG
PL
5' UTR
5' UTR
5' UTR
5' UTR
HN
FG
FG
FG
MG
Toxin
gyrB
GyrB
Average
of
Replicate
CP
Infectious
Units
TCID50/ CFU
or µl of NPA
per Run
12.9
14.2
6.84E+02
14.5
19.8
0.74µl NPA
NA
13.9
12.4
21.6
23.6
7.8
9.2
1.22E+02
0.74µl NPA
1.88E+05
NA
11.5
12.2
13.0
11.8
24.7
27.3
24.4
22.5
24.5
24.2
12.0
15.5
12.4
19.8
12.9
12.7
17.6
23.1
2.16E+04
NA
6.84E+03
0.74µl NPA
7.43E+02
1.22E+05
6.84E+03
1.22E+06
1.17E+01
1.22E+04
1.49E+03
81.49E+03
1.54E+03
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)
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Instrument Automation –
Electro-pneumatic Integration
Silicone bladders
inflate over blisters to
move liquids
Air Channels
Valve
Pistons close channels
to select flow path
All components
integrated in a single
core module
Window - 2nd
Pneumatic Bladders
Stage PCR
10
Prototype FilmArray™ Instrument
Fully automated
1 hour to result
System weight 14 lbs +
laptop
Five prototype systems in
operation
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The Instrument Generates a Complex Data Set
12
MetaAnalysis Summary
13
Analysis for each organism
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Analyze the results for each assay
15
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
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Lackland AFB Demo – Jul 07
3 Pre-production
FilmArray™ Instruments
Pooled ATCC samples and
frozen de-identified
clinical specimens
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Lackland Results
Blinded test demonstrated very reproducible
Drop Page Fields Here
multiplex results across three instruments
Friday Mix 2, 3 runs
SpyoRpob.02
SpnePly.01
RSVMG.01
PIV3FG.01
PIV2FG.02
PIV1HN.01
MpneTox.02
MpneGyrb.01
FluBHA.01
FluAN2.06
FluAN1.11
FluAH3.04
FluAH1.09
2.31E+04
2.16E+05
4.61E+06
6.92E+04
Drop More Series Fields
CoVOC43NG.07
Staphylococcus aureus
HSV-1
Neisseria mucosa
Hemophilus influenzae
Average of Cp
30
25
20
15
10
5
0
CoV229EPL.01
Influenza A H3N2
Influenza B
Adenovirus B & E
Coronavirus 229E
M. pneumoniae
S. pyogenes
Strep. pneumoniae
CEID/TCID/cfu
/run
2.16E+05
6.84E+04
6.84E+03
1.22E+03
1.54E+04
7.54E+07
6.15E+03
AdHex-iF1R2
Organism
Assay
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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
Viruses
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Organisms Tested
FilmArray
Bacteria Assays
3
36
DFA
Viruses
8
19
Detection Rates for FilmArray and DFA
Percent Results/ Sample
60
50
40
FilmArray
30
DFA
20
10
0
Negative
Single
Dual
Triple
20
0
Mycoplasma pneumoniae
Coronavirus SARS
Coronavirus NL63
Coronavirus HKU1
Coronavirus 229E
Coronavirus 0C43
Chlamydia pneumoniae
Bordatella pertussis
Bocavirus
Human Rhinovirus
Parainfluenza Virus 4
Parainfluenza Virus 3
Parainfluenza Virus 2
Parainfluenza Virus 1
Respiratory Syncytial Virus
Influenza B
Influenza A
Human Metapneumovirus
Adenovirus
PCMC Study Results To-date
Organisms ID'd in 143 Samples
35
30
25
20
FilmArray
15
DFA
10
5
21
Triple Infection
Bocavirus – black
PIV2 – blue
Human Rhinovirus - green
Bocavirus – black, PIV2 – blue, Human
Rhinovirus - green
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