plex-id fully automated analysis of pcr products by high performance esi-tof mass spectrometry. a...
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PLEX-IDFully Automated Analysis of PCR Products by High Performance ESI-TOF Mass Spectrometry. A novel Platform for Clinical ResearchMark Van Asten
Diagnostic Technology
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Challenges of Pathogen Detection: Clinical Perspective
Key Question: What is the best treatment?
What organism is causing the infection?
What is its resistance profile?
How virulent is it?
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• Over 1000 agents known to infect humans*– 217 virus species– 538 bacterial species– 307 fungi– 66 parasitic protozoa
• Additional plant and animal pathogens not counted• Potential bio-engineered organisms• Numerous strain variations of each species (i.e., >150 recognized strains of Streptococcus
pyogenes)• Emergence of multi-drug resistant and highly pathogenic strain types• Current molecular platforms are not adequate
Taylor et al, Phil. Trans. R. Soc. Lond. B (2001) 356, 983-989*
Challenges of Pathogen Detection: Laboratory Perspective
There are numerous naturally occurring infectious organisms
This is not a simple question to
answer
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PLEX-ID offers rapid identification without culture
• Designed for broad identification of all microbes– Bacteria, Viruses, Fungi, Parasites
• Detects complex mixtures of microbes
• Direct sample testing
• Capable of providing high resolution genotyping and strain identification
• Enables detection of novel microorganisms
• Provides relative organism abundance
• Detection and identification in 6-8 hrs
A New Technology for Microbial Identification
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Ibis Biosciences
Isis Pharmaceuticals, Carlsbad, CA› Biopharma (Antisense Technology)
Ibis Biosciences, a subsidiary of Isis; founded end of 90s
Projects:- DARPA: Biodefense
- CDC, NIH: Clinical diagnostics and epidemiology- FBI: Human and microbial forensics
Creation of the technology used by the Ibis T5000 Universal Biosensor, a revolutionary approach to identifying infectious disease agents
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Molecular Technologies can Address this Challenge
• PCR– Gold Standard in many Clinical Virology Labs– Limited number of PCR assays for certain common bacteria exist– Typically targets a single organism or a small group of organisms– Not well-suited to comprehensively covering such a large and diverse group
of organisms
• Sequencing– Gold Standard for confirming organism identity– Labor-intensive and time-consuming– Cannot handle complex mixtures of organisms – must use individual isolates
Neither offer the breadth of coverage and/or TAT required
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PLEX-ID is the Molecular Solution to Culture
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TECHNOLOGY OVERVIEW
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What is PCR ESI-MS Technology?
• Coupling of broad PCR amplification with Electrospray Ionization Time-of-flight Mass Spectrometry (ESI–TOF–MS)– Mass of PCR product (amplicon) is accurately determined by mass
spec, converted to a unique base composition, then compared to a database of known targets for identification
• When PCR ESI-MS is applied to microbial identification– Sensitive identification of known and uncharacterized microorganisms
without culture
The Technology behind PLEX-ID is PCR-ESI MS
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PLEX-ID and Front End Processing Plattforms
Precellys 24
Bead Beater
Kingfisher
Sample extraction
EppendorfMastercycler Pro
(3 or 4)
Front End Processing PLEX-ID
EVO75
Kingfisher plate setup / PCR plate setup
Computer Hardware
Workflow Tracking SW
T6000
Desalting
ESI TOF Mass Spec
Computers (2)
Abbott Labeled
Data Connectivity between Workflow Tracking Computer and T6000
No Direct Data Connectivity with Front End Components
Abbott LabeledAbbott Decal upon Installation Abbott Decal upon Installation Abbott Labeled
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Primers bind to conserved regions in ALL
(or broad groups of) bacteria
Foundation of the Technology: Design of PCR Primers Targeted to Universally Conserved Genes
Highly Variable Region
Informative region varies by type of bacteria
Resulting PCR product acts like a “fingerprint” to identify the microbes
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Primers Target Highly Conserved Regions
Broad Primers Covering Bacteria
Primers Covering Proteobacteria
Primers Covering Gamma Proteobacteria
Primers Covering Fusobacteria
Built-in-RedundancyFor MRSA: 8 primer pairs• 4 rRNA • 2 for Fusobacteria• 1 for Staphlococcus• 1 for MecA
Primers Covering Staphlococcus
Primers Covering Antibiotic Resistance
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Identify genomic regions for identification:Variable DNA sequences flanked by conserved sequences
Broad range primerPCR
Broad range primerConserved DNA Variable DNA Conserved DNA
1STEP Amplify nucleic acids to
measure: Use broad-range, unbiased PCR primers
2STEP
Measure nucleic acid:ESI-TOF (Electrospray Ionization Time-of-Flight)3
STEP
Identify the organisms: Base-composition fingerprints4
STEP
As: 17Gs: 30Cs: 11Ts: 61
The Ibis Approach to Pathogen ID and Strain-Typing
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PLEX-ID Process Part 1:Nucleic Extraction and Broad Range PCR
Microbe Mixture
PCR Products
Extract Nucleic Acids
Broad Range Primers
PCR Amplification
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Ibis Process Part 2:MS Analysis and Signal Processing
6 33734.22 A19G21 C17T27
1000
(ESI) (TOF)(ESI) (TOF)
Ion Source Mass Analysis Detector
Generation of Ions Seperation of Ions Detection of Ions
Ion Source Mass Analysis Detector
Generation of Ions Seperation of Ions Detection of Ions
Ion Source Mass Analysis Detector
Generation of Ions Seperation of Ions Detection of Ions
Ion Source Mass Analysis Detector
Generation of Ions Seperation of Ions Detection of Ions
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Converting Masses to Base Composition
800 900 1000 1100 1200 1300m/z
800 900 1000 1100 1200 1300m/z
MW = 32,588.90 amuA28 G29 C25 T24
Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g
A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu
Weight = 4.6 grams 2 dimes
??
.. ... .
Mass spectrum
Scale
.. ... .
Weight = 377.33 g28 Pennies29 Nickels25 Dimes24 Quarters
You can distinguish any change, even a single nucleotide
Each coin has unique weight From weight determine the # of each
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Converting Masses to Base Composition
800 900 1000 1100 1200 1300m/z
800 900 1000 1100 1200 1300m/z
MW = 32,588.90 amuA28 G29 C25 T24
Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g
A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu
Weight = 4.6 grams 2 dimes
??
.. ... .
Mass spectrum
Scale
.. ... .
Weight = 377.33 g28 Pennies29 Nickels25 Dimes24 Quarters
You can distinguish any change, even a single nucleotide
Not for use in diagnostic procedures.
Coins ≈ Nucleotides From weight determine the # of each
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Converting Mass to Base Composition
800 900 1000 1100 1200 1300m/z
800 900 1000 1100 1200 1300m/z
MW = 32,588.90 amuA28 G29 C25 T24
Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g
A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu
Weight = 4.6 grams 2 dimes
??
.. ... .
Mass spectrum
Scale
.. ... .
Forward Reverse Double
Combinations
A28 G29 C25 T24
MW = 32,889.45 Da
MW = 33,071.46 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
A28 G29 C25 T24
MW = 32,889.45 Da
MW = 33,071.46 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
A27 G25 C30 T25
MW = 32,889.45 Da MW = 33,374.26 Da
MW = 33,071.46 Da MW = 37,231.15 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
A G C T27 + 25 + 30 + 25 = 32889.45 Da
A G C T25 + 30 + 25 + 27 = 33071.46 Da
A G C T27 + 25 + 30 + 25 = 33374.26 Da
A G C T25 + 30 + 25 + 27 = 37231.15 Da
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Getting to a Single Base Composition Solution
A28 G29 C25 T24
MW = 32,889.45 Da
MW = 33,071.46 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
A28 G29 C25 T24
MW = 32,889.45 Da
MW = 33,071.46 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
A28 G29 C25 T24
MW = 32,889.45 Da MW = 32,889.45 Da
MW = 33,071.46 Da MW = 33,071.46 Da
A25 G26 C30T25A24 G25 C29 T28A25 G25 C30 T26A24 G27 C31 T28A24 G27 C27 T24
A24 G27 C27 T24A28 G31 C27 T24A26 G30 C25 T25A28 G29 C25 T24A25 G30 C26 T25
A = TC = GT = AG = C
Number of possible base combinations
Highly accurate mass measurement Small amplicon size of 80-150 base pairs Complementarity
800 900 1000 1100 1200 1300m/z
800 900 1000 1100 1200 1300m/z
MW = 32,588.90 amuA28 G29 C25 T24
Penny = 2.500 gNickel = 3.950 gDime = 2.268 gQuarter = 5.670 g
A = 313.0576 amuG = 329.0526 amuC = 289.0464 amuT = 304.0461 amu
Weight = 4.6 grams 2 dimes
??
.. ... .
Mass spectrum
Scale
.. ... .
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Unique ID from Multiple MeasurementsDifferent primer pairs result in PCR products of different mass and
base composition
Organism Mass Base Composition
Escherichia coli 35641.855 A22 G39 C29T25
Escherichia coli 35870.920 A27 G33 C27 T29
Staph aureus 35240.807 A24 G35 C30 T25
Staph aureus 35744.918 A29 G29 C30 T28
Bacillus anthracis 35278.823 A26 C34 C27 T27
Bacillus anthracis 35174.799 A25 C32 C30 T27
PCR 1PCR 2PCR 3PCR 4PCR 5PCR 6PCR 7PCR 8
PCR 1PCR 2PCR 3PCR 4PCR 5PCR 6PCR 7PCR 8
PC
R/T
arg
et
Re
gio
n 1
PC
R/T
arg
et
Re
gio
n 2
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Using Base Composition to Differentiate Microbes
Unknown enterobacteria species
• Base composition cloud from a cluster of enterobacteria
Y.enterocol.
E.coli K12E.coli O157S.typhiS.typhimuriumY.pestis
f1 r1
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Bacterial Tree of Life
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Hospital Organisms
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Yersinia pestis
Clostridium botulinum
Bacillus anthracis
Burkholderia mallei
Biodefense Organisms
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Salmonella enterica
Listeria monocytogenes
Mycobacterium tuberculosis
Bordetella pertussis
Public Health Organisms
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Fungal Identification – Application in Development
• Fungal Coverage is achieved by broad-based rDNA and family specific primer pairs
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PLEX-ID APPLICATIONS
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Basic Functions Performed By PLEX-ID Assays
Broad Identification• Broad
identification of a wide range of microorganisms
Genotyping• Specific typing of
strains or serotypes of a single species
Drug Resistance / Virulence• Independent
markers of resistance and virulence are reported
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Current Applications for the PLEX-ID are Broad and Cross Many Industries
Biological Research Forensic Analysis Biodefense Biopharma Public Health
Microbial Detection Mito typing
STR profiling
Agent detection
Agent identificationQuality Control
Food testing
Water safety testing
Epidemiology