plex-id technology overview/assay descriptions

PLEX-ID Technology Overview/Assay Descriptions

Mike McDermottOklahoma State Dept HealthPublic Health Laboratory

PLEX-ID Instrument Overview

Desalting

ESI Mass Spec

Organism identification

Design Inputs for PLEX-ID• Samples should not wait for

processing• Processing must be flexible

enough to accommodate shifting workflow

• Time to first result (ie. Turn Around Time) should be minimized

• Comprehensive sample trackingo Simplified, easy to interpret

resultso High throughput

Steps in Pathogen Detection with Ibis Technology

PLEX-ID Fundamental DesignAutomate Post PCR Applications

o Desalto ESI-TOF Mass Accumulationo Identification of Amplified Species

• Throughput of up to 100 samples in a 8hr period depending

• Research and Clinically Appropriate System

• On board reagents to process 15 96-well trays

• Two system configurationso Diagnostic o Not for Diagnostic Use

PLEX-ID Diagnostic System Set Up

• PROVIDED• PLEX-ID BB

o Bead Beater-Precellys 24

• PLEX-ID SPo Sample Prep-

Kingfisher Flex• PLEX-ID FH

o Plate Setup- Tecan EVO75

• PLEX-ID TCo Thermal cycling-

Eppendorf

• NOT PROVIDED• Microfuge• Heating block• Incubator• Plate Centrifuge

Plex ID Not-For-Diagnostic-UseSet up

• PROVIDED• PLEX-ID Workstation

Computer• PLEX-ID TC

o Thermal cycling-Eppendorf

• NOT PROVIDED• Microfuge• Heating block• Incubator• Plate Centrifuge• Kingfisher• Bead Beater• Tecan Fluid Handler

Assay Kit Configuration • Process tubes-ordered separately• Sample Extraction consumables and buffers

o Pre-extraction lysis bufferso Extraction lysis, wash, and elution buffers

• PCR bufferso Dilution buffers, Enzyme buffers

• PLEX-ID Analyzero Clean-up reagentso Magnetic bead cartridge and components

OSDH PHL WorkflowSample Extraction

PCR

Specimen Tracking

Pre-extraction

PLEX-ID BB Commodities- Process Tubes

• Necessary for sample tracking

• Come with or without beads

• Ordered separately

PLEX-ID BB• Precellys-24 Bead Beater

o Off the shelf configurationo Branding affixed at

installation

• Bacterial and fungal samples require physical and chemical lysis to improve genomic yield

• Executes sequential bursts within one program.

• Bead Beating 15-60 minutes depending upon number of samples

• Incubate in heat block and spin down

Process Tubes to Carrier

Deep-well plates for Sample Extraction

PLEX-ID Sample preparation for extraction

Transfers samples from tubes to deep wellsample clean-up plates. For flu assay, also fillsthe elution plate.

PLEX-ID FH• EVO-75 fluid

handlero Tecan, Mannedorf,

Switzerlando Abbott labeled

• Key reason for selectiono Service, support, and

spare parts infrastructure

o Abbott experience

PLEX-ID SP Kingfisher• Modified version of Kingfisher

Flex Kingfisher extractoro Identical electronics,

firmware, motion, and heating block

o Magnetic head interchangeability limited to 2 heads

• Magnetic particle separationo lysiso magnetic particle binding

separation and washingo elution steps

• Extraction: PLEX-ID SP: 30 minutes setup plus 30 minutes run time (up to 96 samples)

PLEX-ID FH PCR plate preparation

PLEX-ID TC• Master Cycler ProS

oOff the shelf configuration

o 2 hr DNA PCRo 3 hr RNA PCR

• Control PaneloConnect up to 5

cyclers

PLEX-ID Set-up

PLEX-ID Analyzer start screen

Example Result Report

PCR/ESI-MS NFDU Workflow: Results in a Single 8-Hour Shift

Nucleic Acid Extraction

And PurificationBroad Range PCR Desalting of Amplicons ESI-MS-TOF

Base Composition

Triangulation

10 minutes/well 30 sec/well 1-2 minutes/sample~2 hours

Open method

2-3 hours/plate

DNA: 2 hours per plateRNA viruses: 3 hours per plate

70 min/plate

Desalting is continually performed ahead of MSMS has two alternating ESI probes for increased throughput

Not For Use In Diagnostic Procedures.6-8 hours

PLEX-ID Assay Menu

• Biothreat• B. anthracis GT*

• BAC Detection• Broad Bacterial• Flu Detection• MDR-TB• MRSA• Acinetobacter GT*• Broad Fungal• Broad Viral 1• C difficile*• Group A Strep GT*• Mycoplasma* • Pneumococcus Serotyping*• Respiratory Virus• Vector-Borne*

• Food-Borne Bacteria

Biodefense Routine Monitoring Food Safety

*Assay available 2011

Not For Use In Diagnostic Procedures

PLEX-ID Assays: Sample Type Recommendations


Product Name Preferred Sample Types Satisfactory Sample TypesPLEX-ID Acinetobacter GT Throat & surface swab, Tissue/Cells Nasal swab, water/air filters, body fluids, Foods

PLEX-ID Broad BacteriaPLEX-ID Broad Bacteria LoPLEX-ID BAC Detection

Culture, Water filter, Air filter Surface swab, Body fluids

PLEX-ID Biothreat Culture, Water filter, Air filter Surface swab, Body fluids, Environmental

PLEX-ID Cdiff isolates Surface swab, Water/Air filter, FoodPLEX-ID Flu DetectionPLEX-ID Respiratory Virus

Nasal swab, NPA, Sputum Surface swab, Air/water filter, Body fluids

PLEX-ID Food-Borne Bacteria Enriched culture samples, isolates

PLEX-ID Group A Strep GT Nasal/Throat swab, Tissue/Cells, Sputum, culture Surface swab, Air/water filter, Body fluids

PLEX-ID MDR TB Sputum, Culture Nasal/Throat swabs, Tissue/cells, Body fluids

PLEX-ID mtDNA Buccal swab, Blood, Hair, Bone

PLEX-ID Mycoplasma Culture Media, Cell Culture

PLEX-ID Vector-Borne Tick, mosquito, blood, tissue

PLEX-ID Biothreat Assay• Detection and identification of many important bacterial

and viral biothreat agents• Distinct parsing of 17 targeted agents from

nonpathogenic near neighbors• Identification of near neighbors for reassurance of false

alarm• Supplements LRN assays• Analysis of direct specimens

o Validation with water and air filter sampleso Others are appropriate as well

Wells Per Sample: 16

Primer Pairs Per Sample: 33

Samples Per Kit: 60

Input NA Volume: 5µl/well

PLEX-ID Biothreat Targeted Organisms Bioagent s (or surrogat e) Near Neighbors

1 Bacillus anthracis B. cereu s, B. thu ringiensis, B. myc oides, 10 other bacilli 2 Brucella mel itensis Biovars: abortus, canis, ovis, suis, ma ris, neot omae 3 Burkh olderia pseud omallei, mallei B. cepa ciae, B. fungor um 4 Clostridium botulinum C. acetob utylicum, C. sordelli, C. sporogenes 5 Clostridium perfringens C. acetob utylicum, C. sordelli, C. sporogenes 6 Coxiel la burnetii Legione lla species 7 Francis ella tularensis F. tularensis subspeci es and biovars (over 30) 8 Rickettsia prowazek ii Other Rickettsia (ove r 24 species) 9 Vibrio cholerae Iden tifies V. cholerae O1 & O139 , V. parahaem olyticus, V. vulnificus, 5 othe rs

10 Yersinia pes tis Y. pseudotub erculosis, Y. kristensen ii, Y. enter ocolitica, Y. frederiksenii, Y. ruck eri 11 Esche richia coli O157:H7 E. coli, Shig el la, Salmonel la, Klebsiella, Prote us (over 100 species) 12 Shigel la flexn eri E. coli, Shig el la, Salmonel la, Klebsiella, Prote us (over 100 species) 13 Salmone lla enterica E. coli, Shig el la, Salmonel la, Klebsiella, Prote us (over 100 species) 14 Variola virus ( monkeyp ox) All orthopox viruses, including vaccinia, camelpox, cowpo x, etc. 15 Ebola virus, strain Resto n Marburg virus 16 Avian influenza H 5N1 (Avian H 5N2) Huma n A, B, & C type s, other Avian type s, porcine 17 Venezuela n equine enceph alitis virus WEE, EEE, Chikungu nya virus

Targeted organisms are confidently distinguished from Near Neighbors


PLEX-ID Flu Detection Assay• Identification, characterization, and differentiation of

influenza A, B, and C viruses directly from human, avian, animal, or environmental sampleso Human, avian, equine, swine, etc.o Pandemic H1N1o Seasonal H3N2/H1N1o Newly emerging variants

• H1 & N1 primer pairs specific for 2009 H1N1 added

• N1 region contains the primary Tamiflu SNP (H275Y)9 Gene Targets

Flu-All-PB1FluA-NPFluA-M1FluA-PAFluA-NS1FluA-PB2H1FluB-NPN1-Tamiflu-R

Wells Per Sample: 8


Samples Per Kit: 120



PLEX-ID Flu Detection• Coverage: All known Influenza

A, B, and C viruseso H- and N- typing (outside of

pH1N1) is inferred based upon base count signatures in other segments

• Database:o More than 9800 strains of

Influenza A (will not all be uniquely parsed)

o Influenza Bo Influenza C


Organism Target Segment Plate RowAll Influenza PB1 A

Influenza A NP B

Influenza A M1 C

Influenza A PA D

Influenza A NS1 E

Influenza A PB2 F

Influenza A pH1N1 H1 G

Influenza B NP

HInfluenza A pH1N1 N1

Tamiflu-R*

PLEX-ID Food-Borne Bacteria • Detects and identifies of E. coli, Shigella spp, Listeria spp., and

Salmonella spp.o Detects >250 members of Salmonella enterica species complexo Differentiates 7 major Salmonella enterica subspecies: Type I-VIIo Further differentiates Salmonella enterica subspecies enterica

(Type I) into serotype clusters, for e.g., Newport, enteritidis, typhimurium, typhi, paratyphi, saintpaul, Heidelburg, etc.

o Detects E. coli species & differentiates E. coli O55:H7 and O157:H7 from other E. coli serotypes

o Detects & differentiates Shigella species (boydii, dysenteriae, flexneri and sonnei)

o Detects Listeria species & differentiates Listeria monocytogenesfrom Listeria species (e.g.: innocua, ivanovii, seeligeri, welshimeri)


PLEX-ID Food-Borne Bacteria• Gene Targets

o DNA mismatch repair protein (mutS) o malate dehydrogenase (mdh) o Peptide chain release factor (prfA)o Invasion associated protein (iap)

• Permits analysis of pre-enriched samples – isolates not required


Wells Per Sample: 8


Samples Per Kit: 60


PLEX-ID Food-Borne Bacteria Plate Layout


Workstation Results

PlexID Report

• Influenza Detection RUO Assayo Qiagen extractions o Compared vs. Luminex RVP and CDC rRT-PCR Flu

IVDo Compared to CDC strain typing

• Foodborne Bacterial Detection RUO Assay o Tested approx 40 Salmonella isolates (26 serotypes)o Tested 77 STEC isolates (12 O groups)o GN broths known and unknown STEC +o Evaluated boil prep, Qiacube, EZ1 & Instagene

extraction methods

OSDH PHL Evaluation

• Ease of Use• Shorter turn-around-time compared to culture

o Extraction 30 minutes o Mastermix plate set up template addition 15 minuteso PCR 2-3 hourso PlexID plate reading 58 minutes

• Increased range of detection• Increase sample types• Expands PHL capacity to detect emerging pathogens

Benefits

• Internal Validation• CAP or CLIA compliance• How do you test all of the possible sample types

• Cost • Kit/Reagent configurations

• Maybe too much information• Still needs more bacterial serological data

Challenges

• Rangarajan Sampath, Ph.D.IBIS, Science & Technology Director

• Michael Poynton, Tina Han, Ph.D.Abbott Molecular, Application Specialist

• Rebecca Bell FDA/CFSAN

Acknowledgements

• Mike McDermottMolecular Biology Section LeaderOklahoma State Dept of Health1000 NE 10th

Oklahoma City, OK 73117405 [email protected]

Contact

mailto:[email protected]�

plex-id technology overview/assay descriptions

Documents