PLEX-IDFully 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, 983-989 Challenges of Pathogen Detection: Laboratory Perspective This is not a simple question to answer There are numerous naturally occurring infectious organisms *
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
What is PCR ESI-MS Technology? The Technology behind PLEX-ID is PCR-ESI MS • 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
PLEX-ID and Front End Processing Plattforms Front End Processing PLEX-ID Abbott Decal upon Installation Abbott Labeled Abbott Labeled Abbott Decal upon Installation Abbott Labeled Eppendorf EVO 75 Precellys 24 Kingfisher Mastercycler Pro Kingfisher plate setup / Bead Beater Sample extraction PCR plate setup ( 3 or 4 ) No Direct Data Connectivity with Front End Components T 6000 ESI TOF Data Connectivity between Workflow Tracking Computer and T 6000 Mass Spec Desalting Computers ( 2 ) Computer Hardware Workflow Tracking SW
Foundation of the Technology: Design of PCR Primers Targeted to Universally Conserved Genes Primers bind to conserved regions in ALL (or broad groups of) bacteria 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 Primers Covering Staphlococcus Primers Covering Antibiotic Resistance • Built-in-Redundancy • For MRSA: 8 primer pairs • 4 rRNA • 2 for Fusobacteria • 1 for Staphlococcus • 1 for MecA
The Ibis Approach to Pathogen ID and Strain-Typing Amplify nucleic acids to measure: Use broad-range, unbiased PCR primers STEP STEP STEP STEP 1 3 2 4 Identify the organisms:Base-composition fingerprints Measure nucleic acid:ESI-TOF (Electrospray Ionization Time-of-Flight) As: 17 Gs: 30 Cs: 11 Ts: 61 Identify genomic regions for identification: Variable DNA sequences flanked by conserved sequences PCR Conserved DNA Variable DNA Conserved DNA Broad range primer Broad range primer
PLEX-ID Process Part 1:Nucleic Extraction and Broad Range PCR Extract Nucleic Acids Microbe Mixture Broad Range Primers PCR Amplification PCR Products
Ibis Process Part 2:MS Analysis and Signal Processing 6 33734.22 A19G21 C17T27 1000 15
Converting Masses to Base Composition Weight = 377.33 g 28 Pennies 29 Nickels 25 Dimes 24 Quarters Each coin has unique weight From weight determine the # of each You can distinguish any change, even a single nucleotide
Converting Masses to Base Composition Weight = 377.33 g 28 Pennies 29 Nickels 25 Dimes 24 Quarters Coins ≈ Nucleotides From weight determine the # of each You can distinguish any change, even a single nucleotide Not for use in diagnostic procedures.
Converting Mass to Base Composition A24 G27 C27 T24 A24 G27 C27 T24 A24 G27 C27 T24 MW = 32,889.45 Da MW = 33,374.26 Da MW = 32,889.45 Da MW = 32,889.45 Da A28 G31 C27 T24 A28 G31 C27 T24 A28 G31 C27 T24 A = T A = T A = T + 30 + 30 + 25 + 25 27 27 + 25 + 25 A A G G C C T T = 32889.45 = 33374.26 Da Da A26 G30 C25 T25 A26 G30 C25 T25 A26 G30 C25 T25 A28 G29 C25 T24 A28 G29 C25 T24 A28 G29 C25 T24 C = G C = G C = G A25 G30 C26 T25 A25 G30 C26 T25 A25 G30 C26 T25 A28 G29 C25 T24 A27 G25 C30 T25 A28 G29 C25 T24 T = A T = A T = A A25 G26 C30T25 A25 G26 C30T25 A25 G26 C30T25 A24 G25 C29 T28 A24 G25 C29 T28 A24 G25 C29 T28 + 25 + 25 + 27 + 27 25 25 + 30 + 30 G = C G = C G = C A A G G C C T T = 33071.46 = 37231.15 Da Da A25 G25 C30 T26 A25 G25 C30 T26 A25 G25 C30 T26 A24 G27 C31 T28 A24 G27 C31 T28 A24 G27 C31 T28 A24 G27 C27 T24 A24 G27 C27 T24 A24 G27 C27 T24 MW = 33,071.46 Da MW = 37,231.15 Da MW = 33,071.46 Da MW = 33,071.46 Da Double Forward Reverse Combinations
Getting to a Single Base Composition Solution • Highly accurate mass measurement • Small amplicon size of 80-150 base pairs • Complementarity Number of possible base combinations
Unique ID from Multiple Measurements • Different primer pairs result in PCR products of different mass and base composition Organism Mass Base Composition Bacillus anthracis 35278.823 A26 C34 C27 T27 PCR/Target Region 1 Escherichia coli 35641.855 A22 G39 C29T25 Staph aureus 35240.807 A24 G35 C30 T25 Bacillus anthracis 35174.799 A25 C32 C30 T27 Escherichia coli 35870.920 A27 G33 C27 T29 PCR/Target Region 2 Staph aureus 35744.918 A29 G29 C30 T28
Unknown enterobacteria species Using Base Composition to Differentiate Microbes f1 r1 E.coli K12 • Base composition cloud from a cluster of enterobacteria E.coli O157 S.typhi S.typhimurium Y.pestis Y.enterocol.
Biodefense Organisms Burkholderia mallei Bacillus anthracis Clostridium botulinum Yersinia pestis 24
Public Health Organisms Bordetella pertussis Mycobacterium tuberculosis Listeria monocytogenes Salmonella enterica 25
Fungal Identification – Application in Development • Fungal Coverage is achieved by broad-based rDNA and family specific primer pairs
Current Applications for the PLEX-ID are Broad and Cross Many Industries Biological Research Forensic Analysis Biodefense Biopharma Public Health Food testing Water safety testing Epidemiology Microbial Detection Mito typing STR profiling Agent detection Agent identification Quality Control