plex-id fully automated analysis of pcr products by high performance esi-tof mass spectrometry. a...

PLEX-IDFully Automated Analysis of PCR Products by High Performance ESI-TOF Mass Spectrometry. A novel Platform for Clinical ResearchMark Van Asten

Diagnostic Technology

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?

• 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

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

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

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

PLEX-ID is the Molecular Solution to Culture

TECHNOLOGY OVERVIEW

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

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

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

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

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

PLEX-ID Process Part 1:Nucleic Extraction and Broad Range PCR

Microbe Mixture

PCR Products

Extract Nucleic Acids

Broad Range Primers

PCR Amplification

15

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







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

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




??

.. ... .

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

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




??

.. ... .

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




A27 G25 C30 T25

MW = 32,889.45 Da MW = 33,374.26 Da

MW = 33,071.46 Da MW = 37,231.15 Da




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

Getting to a Single Base Composition Solution

A28 G29 C25 T24

MW = 32,889.45 Da

MW = 33,071.46 Da




A28 G29 C25 T24

MW = 32,889.45 Da

MW = 33,071.46 Da




A28 G29 C25 T24

MW = 32,889.45 Da MW = 32,889.45 Da

MW = 33,071.46 Da MW = 33,071.46 Da




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




??

.. ... .

Mass spectrum

Scale

.. ... .

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

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

Bacterial Tree of Life

Hospital Organisms

24

Yersinia pestis

Clostridium botulinum

Bacillus anthracis

Burkholderia mallei

Biodefense Organisms

25

Salmonella enterica

Listeria monocytogenes

Mycobacterium tuberculosis

Bordetella pertussis

Public Health Organisms

Fungal Identification – Application in Development

• Fungal Coverage is achieved by broad-based rDNA and family specific primer pairs

PLEX-ID APPLICATIONS

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

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

plex-id fully automated analysis of pcr products by high performance esi-tof mass spectrometry. a...

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