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A Complete Solution for Broad Microbial Identification Debbie Boldt-Houle, Ph.D

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A Complete Solution for Broad Microbial Identification Debbie Boldt-Houle, Ph.D A Complete Solution for Broad Microbial Identification Debbie Boldt-Houle, Ph.D. Scientific Affairs Manager Abbott Molecular/Ibis Biosciences Objectives • To gain an understanding of the PLEX-ID and the Ibis Technology • To gain an understanding of the wide range of PLEX-ID assays that are available, including: – Broad Bacteria – Vector-borne – Broad Viral 1 – Broad Fungal Not For Use in Diagnostic Procedures Current Challenges to Microbial Detection: • Over 1000* naturally occurring organisms known to infect humans – 217 virus species – 538 bacterial species – 307 fungi – 66 parasitic protozoa • Numerous strain variations of species (i.e., >300 recognized strains of Streptococcus pyogenes) • Potential bio-engineered organisms • Plant and animal microorganisms * Taylor et al, Phil. Trans. R. Soc. Lond. B (2001) 356, 983-989. Not For Use in Diagnostic Procedures Mainstream Microorganism Detection Today • Culture techniques – Detect a subset of all pathogens – Can take multiple days (weeks) – Many organisms are slow growing or not culturable – False negative results due to conditions • Single agent nucleic acid tests – One test for each microorganism – Need many tests to cover disease state – Fail to detect newly emergent organisms There is currently no good routine method to detect organisms that have never been seen before Not For Use in Diagnostic Procedures PLEX-ID offers a Unique Solution to Microbial Screening & Identification • Broad recognition of microorganisms – Bacteria, Viruses, Fungi, Parasites • Detects mixtures of microbes • High resolution genotyping and strain identification • Enables detection of novel microorganisms • Sensitivity of PCR with relative quantitation • Rapid identification in 6-8 hrs – culture often not required • Throughput up to 100 samples per 8 hours Changes the question “Is this in my sample?” to “What is in my sample?” Not For Use in Diagnostic Procedures Ibis Technology: Unique Approach A Novel Combination of Powerful & Proven Technologies Ribosomal PCR universal primers sensitivity ESI- Mass spectrometry PLEX-ID Triangulation Automated & fast precision specificity Not For Use in Diagnostic Procedures What is the Ibis 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 detection – Sensitive identification of known, unknown, and emerging microorganisms, usually from direct specimens A28 G29 C25 T24 Not For Use in Diagnostic Procedures 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 conserved conserved Informative region varies by type of bacteria (unique) Resulting PCR product acts like a “fingerprint” to identify the microbes Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 1: Nucleic Acid Prep and Broad Range PCR 9 Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 2: MS-ESI Analysis and Signal Processing Spectral signal Generation of ions Separation of ions Detection of ions Mass Detection Signal Processing Masses to Base Compositions Not For Use in Diagnostic Procedures Converting Masses to Base Composition ® 1¢13 5¢15 25¢19 10 20 $7.63 A36 G33 C38 T32 Salmonella enterica Type II salamae Not For Use in Diagnostic Procedures PCR ESI-MS Process Part 3: Triangulation Reconciling Base Counts to Target Identity •Different primer pairs result in PCR products of different mass and base composition for any given microorganism •Database contains over 750,000 base count entries Unique ID from multiple primers Not For Use in Diagnostic Procedures 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 Not For Use in Diagnostic Procedures Resistance Biodefense Organisms Burkholderia mallei Bacillus anthracis Clostridium botulinum Yersinia pestis 14 Not For Use in Diagnostic Procedures Public Health Organisms Bordetella pertussis Mycobacterium tuberculosis Listeria monocytogenes Salmonella enterica 15 Not For Use in Diagnostic Procedures Viral Identification • Viral coverage is achieved with broad-based virus family-specific primer pairs. * *Housekeeping gene: RNA-dependent RNA polymerase Not For Use in Diagnostic Procedures PLEX-ID Technology Features • Suitable for many sample types: – Blood, throat swabs, nasal washes, tissue/cells, culture – Food, water, air, forensic, in-process quality controls • Same experimental process PLEX-ID System for all: Desalting Mass Nucleic Spec Sample acid PCR Extraction Identify target by Convert amplicon comparing base mass to base composition to database composition of >700,000 entries Not For Use in Diagnostic Procedures PCR/ESI-MS Workflow: Results in a Single 8-Hour Shift Nucleic Acid Extraction Base Composition Broad Range PCR Desalting of Amplicons ESI-MS-TOF And Purification Triangulation ~2 hours 2-3 hours/plate 10 minutes/well 30 sec/well 1-2 minutes/sample Open method DNA: 70 min/plate 2 hours per plate RNA viruses: Desalting is continually performed ahead of MS 3 hours per plate MS has two alternating ESI probes for increased throughput 6-8 hours Not For Use in Diagnostic Procedures PLEX-ID using Ibis Technology Assay Capability Broad bacterial or viral Unknown Sample identification Genus/species confirmation Species-level organism identification Drug resistance virulence markers Genotyping MLST/SNP/VNTR High-resolution molecular genotyping Not For Use in Diagnostic Procedures PLEX-ID Crossing Broad Application Boundaries PLEX-ID Biosecurity Bioresearch Food Safety • Biodefense Bacterial & • Flu Detection • Food-Borne Bacteria 1 Viral Surveillance • Broad Bacteria Detection (E. coli, Shigella, Salmonella, Bacterial agents: B. anthracis, • MRSA Genotyping Listeria) B. melitensis, B. mallei / B. • Broad Viral pseudomallei, C. botulinum, C. perfringens, C. burnetii, F. • Respiratory Virus tularensis, R. prowazekii, V. • Broad Fungal cholera, Y. pestis, E. coli O157:H7, S. flexneri, S. • Vector-borne Disease enterica • MDR TB • Pneumococcus Serotyping Viral agents: Variola virus, Ebola • C. Difficile virus, Influenza A virus H5N1, Venezuelan equine encephalitis virus 1 2011 release 20 Not For Use in Diagnostic Procedures PLEX-ID BROAD BACTERIA ASSAY PLEX-ID BROAD BACTERIA LO ASSAY Not For Use in Diagnostic Procedures PLEX-ID Broad Bacteria / Broad Bacteria Lo Coverage and Targets • Designed to detect & identify wide Coverage Target range of bacteria without a priori Broad Bacterial 16S rRNA (4) knowledge Broad Bacterial 23S rRNA (2) Broad Bacterial rpoC • Theoretically covers all major Gram + Firmicutes rplB (2) Bacteria, including intracellular Staphylococcus tufB organisms like Mycoplasma, speciation Chlamydia, and Rickettsia Gram+ Bacilli infB Bacillus speciation sspE • Database currently contains more Gram- Enterobacteria valS than 5000 species base composition Gram- Gamma rpoB entries Proteobacteria Gram- rpoC Betaproteobacteria Gram- Beta and rpoB Gamma Proteobacteria Not For Use in Diagnostic Procedures Broad Bacteria Assay layout • 16 well assay with 16 16s rRNA Gram+ Fimicutes primer pairs per sample 16s rRNA Gram- Enterobacteria • Unknown detections 16s rRNA Gram-Gammaproteobact. will be reported and linked to the closest 23s rRNA 23s rRNA match Gram+ Bacilli 16s rRNA Broad Bacterial Gram-Betaproteobact. Bacillus Gram-Beta/Gammaproteobact. Gram+ Fimicutes Staphylococcus Not For Use in Diagnostic Procedures Spectrum of one broad-range PCR well Calibrant Peaks calculated as mean (A28C28G20T18) of the whole spectrum 16s rRNA primer 346 (from all stages of charge) Lactococcus 4000 lactis locus (A29C28G26T16) Lactococcus lactis locus 2000 (A30C29G26T14) Amplitude 0 28.5 30.25 31 molecular mass (kDa) Not For Use in Diagnostic Procedures Multiple detections in one broad-range PCR well 16s rRNA primer Peaks calculated as mean 3000 from the whole spectrum S. aureus (from all stages of charge) (A30G29C30T29) K. pneumoniae 1500 (A26G32C28T30) Amplitude E. coli (A27G33C27T29) 0 35.6 36.1 36.6 molecular mass (kDa) Not For Use in Diagnostic Procedures PCR/ESI-MS Broad Bacteria / Broad Bacteria Lo Data Usefulness of multilocus polymerase chain reaction followed by electrospray ionization mass spectrometry to identify a diverse panel of bacterial isolates* • 156 blinded bacterial isolates analyzed in the study – 142 (91%) were correctly identified to the genus level – 115 (74%) were correctly identified to the genus and species level – 14 (9%) isolates were misidentified • Misidentification generally due to missing database entries “…Interestingly, based on the PCR/ESI-MS data, the system identified Campylobacter jejuni (ATCC 33560) as Streptococcus parasanguinis, which was confirmed by 16S rRNA PCR and sequencing. This is consistent with other data on several isolates obtained ATCC independently, suggesting at least certain lots of the bacteria could have been mislabeled.” *Baldwin C, et al. Diagn Microbiol Infect Dis. 2009. 63(4): 403-408. Not For Use in Diagnostic Procedures PCR/ESI-MS Broad Bacteria / Broad Bacteria Lo Data Identification of Bacterial Plant Pathogens Using Multilocus Polymerase Chain Reaction/Electrospray Ionization-Mass
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