Next Generation Molecular Testing: PCR/ESI-MS Vanessa Harpin Sr Product Manager Abbott Molecular Not For Use In Diagnostic Procedures.

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

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 Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

Recent news On Sept 14 2009, Abbott’s Ibis Biosciences is awarded the Wall Street Journal Gold award for innovation Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

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. Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

PCR/ESI-MS Workflow Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

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. Not For Use In Diagnostic Procedures. 12 12

PCR/ESI-MS Application Areas Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures. 14

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 Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

Technology Comparison Not For Use In Diagnostic Procedures. 17

Primer Design and Bioinformatics Not For Use In Diagnostic Procedures.

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. Not For Use In Diagnostic Procedures. 19 19

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 Not For Use In Diagnostic Procedures. 20 20

PCR ESI-MS Process Part 2: MS Analysis and Signal Processing 6 33734.22 A19G21 C17T27 1000 21 Not For Use In Diagnostic Procedures. 21

Converting Mass to Base Composition Forward Reverse Double Combinations A28 G29 C25 T24 MW = 32,889.45 Da MW = 33,071.46 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 = 33,374.26 Da MW = 37,231.15 Da A G C T 27 + 25 + 30 = 32889.45 Da 25 + 27 = 33071.46 = 33374.26 = 37231.15 Not For Use In Diagnostic Procedures. 22

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

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 Not For Use In Diagnostic Procedures. 24

The PLEX-ID Platform Not For Use In Diagnostic Procedures.

PLEX-ID Platform Ibis T5000 Not For Use In Diagnostic Procedures.

PLEX-ID Features Capacity Integrated Single power cord into wall jack 33” Capacity 15 - 96 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” Not For Use In Diagnostic Procedures.

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. Not For Use In Diagnostic Procedures.

Food-Borne Bacteria Example If Salmonella is present in a sample, what happens? Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

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? Not For Use In Diagnostic Procedures.

Salmonella Reference Isolate Signatures

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 Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

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 Not For Use In Diagnostic Procedures.

PCR/ESI-MS Results Simple sample-by-sample display More details (basecounts, mass spectra) available Not For Use In Diagnostic Procedures.

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? Not For Use In Diagnostic Procedures.

Contact Information Primary POC: Greg Eppink, Business Development Manager gregory.eppink@abbott.com (419)345-8598 My information: Vanessa Harpin Vanessa.harpin@abbott.com (760)476-3215

Thank You For Your Time! Not For Use In Diagnostic Procedures.