PLEX-ID Fully Automated Analysis of PCR Products by High Performance ESI-TOF Mass Spectrometry. A novel Platform for Clinical Research Mark 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, * 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 Precellys24 Bead Beater Kingfisher Sample extraction Eppendorf Mastercycler Pro (3or4) 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 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-Redundancy For 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 primer PCR Broad range primer Conserved DNAVariable DNAConserved DNA 1 STEP Amplify nucleic acids to measure: Use broad-range, unbiased PCR primers 2 STEP Measure nucleic acid: ESI-TOF (Electrospray Ionization Time-of-Flight) 3 STEP Identify the organisms: Base-composition fingerprints 4 STEP As: 17 Gs: 30 Cs: 11 Ts: 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 A 19 G 21 C 17 T

Converting Masses to Base Composition Weight = g 28 Pennies 29 Nickels 25 Dimes 24 Quarters You can distinguish any change, even a single nucleotide Each coin has unique weightFrom weight determine the # of each

Converting Masses to Base Composition Weight = g 28 Pennies 29 Nickels 25 Dimes 24 Quarters You can distinguish any change, even a single nucleotide Not for use in diagnostic procedures. Coins ≈ NucleotidesFrom weight determine the # of each

Converting Mass to Base Composition Forward Reverse Double Combinations A28 G29 C25 T24 MW = 32, Da MW = 33, Da A25 G26 C30T25 A24 G25 C29 T28 A25 G25 C30 T26 A24 G27 C31 T28 A24 G27 C27 T24 A28 G31 C27 T24 A26 G30 C25 T25 A28 G29 C25 T24 A25 G30 C26 T25 A = T C = G T = A G = C A28 G29 C25 T24 MW = 32, Da MW = 33, Da A25 G26 C30T25 A24 G25 C29 T28 A25 G25 C30 T26 A24 G27 C31 T28 A24 G27 C27 T24 A28 G31 C27 T24 A26 G30 C25 T25 A28 G29 C25 T24 A25 G30 C26 T25 A = T C = G T = A G = C A27 G25 C30 T25 MW = 32, DaMW = 33, Da MW = 33, DaMW = 37, Da A25 G26 C30T25 A24 G25 C29 T28 A25 G25 C30 T26 A24 G27 C31 T28 A24 G27 C27 T24 A28 G31 C27 T24 A26 G30 C25 T25 A28 G29 C25 T24 A25 G30 C26 T25 A = T C = G T = A G = C AGCT = Da AGCT = Da AGCT = Da AGCT = Da

Getting to a Single Base Composition Solution Number of possible base combinations  Highly accurate mass measurement  Small amplicon size of base pairs  Complementarity

Unique ID from Multiple Measurements Different primer pairs result in PCR products of different mass and base composition OrganismMassBase Composition Escherichia coli A22 G39 C29T25 Escherichia coli A27 G33 C27 T29 Staph aureus A24 G35 C30 T25 Staph aureus A29 G29 C30 T28 Bacillus anthracis A26 C34 C27 T27 Bacillus anthracis A25 C32 C30 T27 PCR/Target Region 1 PCR/Target Region 2

Using Base Composition to Differentiate Microbes Unknown enterobacteria species Base composition cloud from a cluster of enterobacteria Y.enterocol. E.coli K12 E.coli O157 S.typhi S.typhimurium Y.pestis f1f1 r1r1

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 ResearchForensic AnalysisBiodefenseBiopharmaPublic Health Microbial DetectionMito typing STR profiling Agent detection Agent identification Quality Control Food testing Water safety testing Epidemiology