1. Next Generation Molecular Testing: PCR/ESI-MS
Vanessa Harpin
Sr Product Manager
Abbott Molecular
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2. Introduction Vanessa Harpin Abbott Molecular
BS Biochemistry, Virginia Tech
PgD Infectious Diseases, London School of Hygiene and Tropical Medicine
Sr Product Manager (Global)
Primary Focus of Food Safety Testing
Abbott Molecular
Division of Abbott
Real-time PCR (m2000), FISH (Vysis)
Acquired Ibis Biosciences and PCR/Mass Spec technology in 2009

3. PCR/ESI-MS Technology
What is PCR/ESI-MS?
Broad, end-point PCR + Electrospray Ionization Time-Of-Flight (ESI-TOF) Mass Spectrometry
Weighing PCR amplicons to determine mass
Using mass to determine the unique base composition (#A,G,C, and T)
Comparing base composition against a database of known organisms
Again, you do not need to test specifically FOR MRSA, C. diff, Avian flu vs. H3N2. The system is inherently designed to be flexible.
PCR/ESI-MS is applied to microbial identification
Capable of rapid and sensitive identification of known, unknown, and emerging microorganisms, often from direct specimens
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4. The Power of PCR/MS Began >20 Years Ago
PCR (Polymerase Chain Reaction)
1983 first used by Kary Mullis
1993 Nobel Prize in Chemistry
Electro-spray Mass Mass Spectometry
1988 first used by John Fenn
2002 Nobel Prize in Chemistry
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5. Recent news
On Sept , Abbott’s Ibis Biosciences is awarded the Wall Street Journal Gold award for innovation
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6. Ibis Biosciences Origins
Originally a subsidiary of Isis Pharmaceuticals
Defense Advanced Research Projects Agency (DARPA) contract in 1997 for Bio-defense
DARPA is the central research and development organization for the U.S. Department of Defense (DoD)
DARPA’s Mission: Provide radical innovation for national security
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7. Ibis Program U.S. Government Partners
Defense Advanced Research Projects Agency (DARPA) funding >6 years for biological weapons defense and troop health and readiness (1997).
Center for Disease Control (CDC) funding initiated 2003 for emerging infectious disease and public health surveillance.
National Institute of Health (NIH) funding initiated 2004 for clinical diagnostics.
Department of Homeland Security (DHS) funding initiated 2004 for microbial forensics and bio-security.
Technology transitions to CDC, US Navy, USAMRIID, Dept. of Homeland Security, FDA and FBI.
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8. Challenges in Microbial Characterization for Food Matrices
Diverse
Complex microbial background
Culture
Takes significant time
Requires specific conditions
Results initially in only basic identification
Typing
Often requires isolate (PFGE, MLVA, serology)
May be limited to previously “known” types
Can be labor intensive and costly
Interpretation can be subjective with some methods
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9. PCR/ESI-MS Technology Capabilities
Broad identification without probes
Applicable to Bacteria, Viruses, Fungi, Parasites
Rapid results – culture often not required
Sensitivity of PCR
Mixtures of microbes can be detected
Genotyping and strain identification
Drug resistance testing without culture
Relative quantification (genome copies)
Direct sample
(or enriched)
Extraction & PCR
Mass Spec
The PCR ESI-MS T5000 Biosensor System takes standard polymerase chain reaction (PCR) products, which are produced using PCR ESI-MS assay kits, cleans the amplicons, and measures their masses on a microTOF mass spectrometer. These masses are used to link the amplicons to specific organisms or markers.
Result
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10. PCR/ESI-MS Workflow
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11. PCR/ESI-MS Workflow Not For Use In Diagnostic Procedures. ~2 hours
Open method
2-3 hours/plate
DNA:
2 hours per plate
RNA viruses:
3 hours per plate
10 minutes/well
30 sec/well
1-2 minutes/sample
70 min/plate
Desalting is continually performed ahead of MS
MS has two alternating ESI probes for increased throughput
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12. PCR/ESI-MS Process Not For Use In Diagnostic Procedures.
(Step 1) The foundation of the PCR ESI-MS Technology is in the selection of the genomic regions used for microbial identification. Broad-based amplification primers are targeted to highly conserved regions of the genome that flank a variable region which is informative for a specific microbe.
(Step 2) Nucleic acids are amplified by these primers to yield an amplicon that is used to identify the organism.
(Step 3) The mass, or weight, of the amplicon is measured by electrospray ionization time-of-flight mass spectrometry (ESI-TOF).
(Step 4) The weight of the amplicon is converted to a base composition – the number of As, Gs, Cs, and Ts – and this information serves as a fingerprint to identify the organism by comparison to a database.
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13. PCR/ESI-MS Application Areas
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14. Basic Design Functions of the 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
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15. PLEX-ID Technology Applications
Food Safety
Biodefense
Bioresearch
Biothreat
Acinetobacter GT
BAC Detection (Bacteria, Antimicrobial Resistance, and Candida)
Broad Bacteria
Flu Detection
Group A Strep GT
MRSA
Mycoplasma
MDR TB
Respiratory Virus
Broad Viral
Vector-Borne (Bacteria, Flaviviruses, and Babesia)
Broad Enteric*
Enteric Toxin Detection*
Food-Borne Bacteria
(E. coli, Shigella, Salmonella, Listeria)
* In Development
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16. Select Research Articles
High-resolution genotyping of Campylobacter species
Identification of pathogenic Vibrio Species in Aquatic Environments
Identification of Bacterial Plant Pathogens
Emerging Influenza Genotypes
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17. Technology Comparison
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18. Primer Design and Bioinformatics
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19. Primers bind to conserved regions in bacteria, viruses, or fungi
PCR/ESI-MS Part 1: Primers Seek Highly Conserved Genes
Primers bind to conserved regions in bacteria, viruses, or fungi
With our expertise in RNA computational genomics, we have examined the genetic sequences from thousands of bacteria and aligned the sequences in a way that enables us to compare sequences across bacteria. In doing so, we’ve identified sequences that are the same, or conserved, across all bacteria. We used our algorithms to scan these alignments for regions like the one shown in this figure, which has the properties that we need for universal bacterial identification. What we show here is a region where there are two universally conserved regions not too far apart from each other, but where the nucleotides in between the conserved regions are variable and differ with each unique species.
The conserved regions serve as anchors for binding our primers, which are modified with Isis’ novel, proprietary chemistries. These modified oligonucleotides enables us to prime from a much broader group of organisms than would otherwise be possible. So we have created primers with a very broad range of detection… this is a dramatic advance over existing techniques.
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20. Highly Variable Region
PCR/ESI-MS Part 1: Primers Seek Highly Conserved Genes
Primers bind to highly conserved regions across all bacteria
Highly Variable Region
With our expertise in RNA computational genomics, we have examined the genetic sequences from thousands of bacteria and aligned the sequences in a way that enables us to compare sequences across bacteria. In doing so, we’ve identified sequences that are the same, or conserved, across all bacteria. We used our algorithms to scan these alignments for regions like the one shown in this figure, which has the properties that we need for universal bacterial identification. What we show here is a region where there are two universally conserved regions not too far apart from each other, but where the nucleotides in between the conserved regions are variable and differ with each unique species.
The conserved regions serve as anchors for binding our primers, which are modified with Isis’ novel, proprietary chemistries. These modified oligonucleotides enables us to prime from a much broader group of organisms than would otherwise be possible. So we have created primers with a very broad range of detection… this is a dramatic advance over existing techniques.
Informative region varies by organism
Resulting amplicon provides a mass signature that identifies microbes
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21. PCR ESI-MS Process Part 2: MS Analysis and Signal Processing
A19G21 C17T 21
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22. 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
A25 G30 C26 T25
A = T
C = G
T = A
G = C
A27 G25 C30 T25
MW = 33, Da
MW = 37, Da A G C T 27
+ 25
+ 30 = Da 25
+ 27 = = =
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23. Primers Provide Varying Levels of Coverage
Broad Primers Covering Bacteria
Primers Covering Proteobacteria
Primers Covering Gamma Proteobacteria
Primers Covering Fusobacteria
Primers Covering Staphlococcus
Broad-based primers (shown at the top left) amplify all species of bacteria. Primers located in different colored regions are more specific, and amplify only those bacteria within the same colored area. In the bacteria assay, the final identification would be confirmed by triangulating information from all the primers used in the assay.
Primers Covering Antibiotic Resistance 23

24. PCR ESI-MS Process Part 3: Triangulation
Triangulation combines the detections from each primer pair into a confident organism call 2 1
As I mentioned before, we do not examine a single locus on the organism’s genome to determine the identity. Instead, we use primers targeted at several different regions to ensure a very precise and confident organism ID. The process of tying together the information from each of the primer pairs used to analyze a sample is called triangulation. This can be done even in the case of mixtures of several microorganisms. 4 3
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25. The PLEX-ID Platform
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26. PLEX-ID Platform
Ibis T5000
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27. PLEX-ID Features Capacity Integrated Single power cord into wall jack
33”
Capacity
well plates
Stat tray for priority samples
Integrated
Desalting unit
Using magnetic beads
Gas generation system for MS
UPS power backup
PCs (2)
Touch screen
Printer
Barcode readers
Single power cord into wall jack
240V / 30A
Process up to 300 samples in 24 hours
85”
82”
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28. PCR/ESI-MS Technology Applied Food Safety
Designed to detect E. coli, Salmonella, Shigella, and Listeria species from enriched samples or isolates
Subtyping of S. enterica
Parsing into serovar groups
Identification of E. coli
Differentiates O157:H7 and O55:H7 from other types
Differentiates the four main Shigella species
S. flexneri, S. boydi, S. dysenteriae and S. sonnei
Identifies presence of Listeria
Differentiates Listeria monocytogenes from L. innocua, and other Listeria spp.
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29. Food-Borne Bacteria Example
If Salmonella is present in a sample, what happens?
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30. Food-Borne Bacteria Example
The mdh and mutS primer pairs will amplify the Salmonella genome during PCR
When the resulting PCR products are analyzed on PLEX-ID, base compositions will be observed for these 6 primer pairs
Subtle differences in the targeted regions will be visible in the base composition signatures
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31. Food-Borne Bacteria Example
Primer Pair 1
Primer Pair 2
Primer Pair 3
Primer Pair 4
Primer Pair 5
Primer Pair 6
We have analyzed the sample with mass spectrometry
We have amplicon basecounts for each of the primer pairs
Now what?
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32. Salmonella Reference Isolate Signatures

33. Salmonella Reference Isolate Signatures
Primer Pair 1
Primer Pair 2
Primer Pair 3
Primer Pair 4
Primer Pair 5
Primer Pair 6
The 6 primer pairs all produce base compositions that can be used to distinguish subspecies and serovars
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34. Food-Borne Bacteria Example
Primer Pair 1
Primer Pair 2
Primer Pair 3
Primer Pair 4
Primer Pair 5
Primer Pair 6
Primer Pair 1
Primer Pair 2
Primer Pair 3
Primer Pair 4
Primer Pair 5
Primer Pair 6
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35. PLEX-ID Food-Borne Bacteria Example
Primer Pair 1
Primer Pair 2
Primer Pair 3
Primer Pair 4
Primer Pair 5
Primer Pair 6
Salmonella subtyping in 6-8 hours
from enriched sample
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36. PCR/ESI-MS Results Simple sample-by-sample display
More details (basecounts, mass spectra) available
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37. PCR/ESI-MS and Food Safety
Other applications?
More granular typing
E. coli/STEC
Salmonella
Other bacteria
Campylobacter
Clostridium
Antibiotic resistance markers
Biothreat, Influenza?
What are your needs and challenges?
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38. Contact Information
Primary POC:
Greg Eppink, Business Development Manager
(419)
My information:
Vanessa Harpin
(760)

39. Thank You For Your Time!
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