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Next Generation Molecular Testing: PCR/ESI-MS

Next Generation Molecular Testing: PCR/ESI-MS. Vanessa Harpin Sr Product Manager Abbott Molecular. Not For Use In Diagnostic Procedures. Introduction. Vanessa Harpin BS Biochemistry, Virginia Tech PgD Infectious Diseases, London School of Hygiene and Tropical Medicine

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Next Generation Molecular Testing: PCR/ESI-MS

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

  2. Introduction • Vanessa Harpin • 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? • Capable of rapid and sensitive identification of known, unknown, and emerging microorganisms, often from direct specimens • 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 PCR/ESI-MS is applied to microbial identification Not For Use In Diagnostic Procedures.

  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 Not For Use In Diagnostic Procedures.

  5. 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.

  6. Ibis Biosciences Origins Ibis Biosciences 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.

  7. 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. Ibis Program U.S. Government Partners Not For Use In Diagnostic Procedures.

  8. Challenges in Microbial Characterization for Food Matrices • 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.

  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 Result Not For Use In Diagnostic Procedures.

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

  11. ~2 hours 2-3 hours/plate 10 minutes/well 30 sec/well 1-2 minutes/sample Open method DNA: 2 hours per plate RNA viruses: 3 hours per plate 70 min/plate Desalting is continually performed ahead of MS MS has two alternating ESI probes for increased throughput PCR/ESI-MS Workflow Not For Use In Diagnostic Procedures.

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

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

  14. Basic Design Functions of the PLEX-ID Assays Not For Use In Diagnostic Procedures.

  15. Food Safety PLEX-ID Technology Applications PLEX-ID Biodefense Bioresearch • Biothreat • Food-Borne Bacteria • (E. coli, Shigella, Salmonella, Listeria) • 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* * In Development Not For Use In Diagnostic Procedures.

  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 Not For Use In Diagnostic Procedures.

  17. Technology Comparison Not For Use In Diagnostic Procedures.

  18. Primer Design and Bioinformatics Not For Use In Diagnostic Procedures.

  19. PCR/ESI-MS Part 1: Primers Seek Highly Conserved Genes Primers bind to conserved regions in bacteria, viruses, or fungi Not For Use In Diagnostic Procedures.

  20. PCR/ESI-MS Part 1: Primers Seek Highly Conserved Genes Primers bind to highly conserved regions across all bacteria Highly Variable Region Informative region varies by organism Resulting amplicon provides a mass signature that identifies microbes Not For Use In Diagnostic Procedures.

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

  22. 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 = 33374.26 = 32889.45 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 A28 G29 C25 T24 A27 G25 C30 T25 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 = 37231.15 = 33071.46 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 = 33,071.46 Da MW = 37,231.15 Da MW = 33,071.46 Da Double Forward Reverse Combinations Not For Use In Diagnostic Procedures.

  23. Primers Provide Varying Levels of Coverage Broad Primers Covering Bacteria Primers Covering Proteobacteria Primers Covering Gamma Proteobacteria Primers Covering Fusobacteria Primers Covering Staphlococcus Primers Covering Antibiotic Resistance

  24. PCR ESI-MS Process Part 3: Triangulation 1 2 4 3 Triangulation combines the detections from each primer pair into a confident organism call Not For Use In Diagnostic Procedures.

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

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

  27. PLEX-ID Features 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.

  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. Not For Use In Diagnostic Procedures.

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

  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 Not For Use In Diagnostic Procedures.

  31. Food-Borne Bacteria Example • We have analyzed the sample with mass spectrometry • We have amplicon basecounts for each of the primer pairs • Now what? Primer Pair 1 Primer Pair 4 Primer Pair 5 Primer Pair 6 Primer Pair 2 Primer Pair 3 Not For Use In Diagnostic Procedures.

  32. Salmonella Reference Isolate Signatures

  33. Salmonella Reference Isolate Signatures Primer Pair 2 Primer Pair 1 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.

  34. Food-Borne Bacteria Example Primer Pair 1 Primer Pair 4 Primer Pair 5 Primer Pair 6 Primer Pair 2 Primer Pair 3 Primer Pair 2 Primer Pair 3 Primer Pair 4 Primer Pair 5 Primer Pair 6 Primer Pair 1 Not For Use In Diagnostic Procedures.

  35. PLEX-ID Food-Borne Bacteria Example Primer Pair 1 Primer Pair 6 Primer Pair 2 Primer Pair 3 Primer Pair 4 Primer Pair 5 Salmonella subtyping in 6-8 hours from enriched sample Not For Use In Diagnostic Procedures.

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

  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? Not For Use In Diagnostic Procedures.

  38. 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

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

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