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New Technologies in Food New Technologies in Food TestingTestingYvonne Hale MSYvonne Hale MS
Florida Department of Agriculture & Consumer ServicesFlorida Department of Agriculture & Consumer ServicesBureau of Food LaboratoriesBureau of Food Laboratories
Tallahassee, FLTallahassee, FLJune 27, 2005June 27, 2005
Why do we need new technology?Why do we need new technology?
Regulatory food testing:Regulatory food testing:Requires confirmed resultsRequires confirmed resultsRequires viable organisms (for example, PFGE)Requires viable organisms (for example, PFGE)–– Nonviable bacteria/viruses not hazardous, except toxinsNonviable bacteria/viruses not hazardous, except toxins–– Sometimes need bacterial counts (MPN)Sometimes need bacterial counts (MPN)
Requires sensitive methodsRequires sensitive methods–– Traditional methods require enrichmentTraditional methods require enrichment–– Stressed organisms, overgrowth, low numbersStressed organisms, overgrowth, low numbers
Requires rapid methodsRequires rapid methods–– Traditional methods have builtTraditional methods have built--in delays due to enrichment, in delays due to enrichment,
growth requirements, ID methodsgrowth requirements, ID methodsRequires testing of high numbers of samplesRequires testing of high numbers of samples
Bioterrorism Bioterrorism demandsdemands rapid, sensitive, specific testing rapid, sensitive, specific testing
OverviewOverview
ImmunomagneticImmunomagnetic Separation (IMS)Separation (IMS)Magnetic immunoassay/concentrationMagnetic immunoassay/concentrationChromogenicChromogenic agaragarPCRPCRRealReal--time PCR/molecular beaconstime PCR/molecular beaconsElectrochemiluminescenceElectrochemiluminescenceRealReal--time PCRtime PCR
ImmunomagneticImmunomagneticSeparation (IMS)Separation (IMS)
OverviewOverview
The IMS PrincipleThe IMS Principle
Uniform, superparamagnetic, monodisperse, Uniform, superparamagnetic, monodisperse, polymer beads coated with a ligand directed polymer beads coated with a ligand directed against the specified target, e.g antibody against the specified target, e.g antibody against surface antigen.against surface antigen.Dynabeads will bind to the target, making it Dynabeads will bind to the target, making it ““magneticmagnetic””, forming a target, forming a target--bead complex. bead complex. Target is then easily isolated from the sample Target is then easily isolated from the sample using a magnet particle concentrator (MPC).using a magnet particle concentrator (MPC).
Concept of IMSConcept of IMS
Y
PCR
YELISA
Plating
OtherMethods
Flexibility of IMSFlexibility of IMSAny sample matrix provided sample Any sample matrix provided sample prepre--enrichment is done enrichment is done ((Food,Water,FecalFood,Water,Fecal material,Blood,Soilmaterial,Blood,Soil))Any detection system Any detection system ((Culture media, PCR, ELISA, ATP, Culture media, PCR, ELISA, ATP, LuminometryLuminometry, Microscopy), Microscopy)
Advantages of Bacterial IMSAdvantages of Bacterial IMS
Increased recovery rateIncreased recovery rateIsolation and concentrationIsolation and concentrationFlexibleFlexibleVersatileVersatileInexpensiveInexpensiveSimpleSimpleReliable Reliable Minimum space requirementsMinimum space requirements
IMS Bacterial Microbiology IMS Bacterial Microbiology ProductsProducts
Dynabeads antiDynabeads anti--Salmonella Salmonella –– Approvals: AOAC, NMKLApprovals: AOAC, NMKL
Dynabeads antiDynabeads anti--E.coli O157 E.coli O157 –– Approvals: ISO, FDAApprovals: ISO, FDA-- BAM, DIN, CCAM, JHM, NMKL, BAM, DIN, CCAM, JHM, NMKL,
U.S.D.A.U.S.D.A.--FSISFSIS
Dynabeads EPEC/VTEC (O145, Dynabeads EPEC/VTEC (O145, O111, O103, O26)O111, O103, O26)–– Products for rapid selective concentration of important Products for rapid selective concentration of important
emerging human pathogens. Difficult to detect strains emerging human pathogens. Difficult to detect strains as they lack phenotypic characteristics, unlike O157as they lack phenotypic characteristics, unlike O157
Dynabeads antiDynabeads anti--ListeriaListeria
Selective Enrichment/Concentration Selective Enrichment/Concentration of Bacterial Pathogensof Bacterial Pathogens
Pre-enrichment of Sample
(6-24 h, 30-42oC)
Selective Enrichment
(18-48 h)
Detection
5min - 24h
IMS
30 min, 18-28oC
Bacterial Manual IMS Bacterial Manual IMS ProcedureProcedure
Sample preSample pre--treatment (enrichment)treatment (enrichment)Add 1mL aliquot of preAdd 1mL aliquot of pre--enriched sample to enriched sample to 20uL Dynabeads20uL Dynabeadsin a microcentifuge tube.in a microcentifuge tube.Incubate for 10 minutes at room temperature using a Incubate for 10 minutes at room temperature using a Dynal Dynal Sample Mixer.Sample Mixer.Perform immunomagnetic separation using the Perform immunomagnetic separation using the MPCMPC--SS..Aspirate supernatant and wash with PBS Tween buffer. Aspirate supernatant and wash with PBS Tween buffer. ReRe--suspend in 100uL of wash buffer and then complete end suspend in 100uL of wash buffer and then complete end detection.detection.
Pre-enrichment → Immunocapture → Separation → Wash andConcentration →Detection
Plating
ELISA
PCR
Other methods
Automated IMS (AIMS)Dynal BeadRetriever
All steps are automated. Simply load and go.All steps are automated. Simply load and go.PrePre--programmed to work with all the Bacterial programmed to work with all the Bacterial Microbiology Dynabeads kits currently available.Microbiology Dynabeads kits currently available.Processes 15 samples within 25 minutes.Processes 15 samples within 25 minutes.Completely closed system without aerosol Completely closed system without aerosol formation.formation.Utilizes inverse IMS with increased bead Utilizes inverse IMS with increased bead recovery & reduced contamination of culture recovery & reduced contamination of culture plates.plates.Continuous operation at room temperature.Continuous operation at room temperature.Small footprint for unit and consumables.Small footprint for unit and consumables.
Advantages of BeadRetriever Advantages of BeadRetriever UnitUnit
MinimizesMinimizes::–– Biological hazards Biological hazards –– HandsHands--on timeon time–– Technician errorTechnician error–– Physical resources Physical resources
PreventsPrevents::–– CrossCross--
contamination contamination –– Background floraBackground flora
IncreasesIncreases::–– SafetySafety–– ThroughputThroughput–– Ease of useEase of use
High fat, high High fat, high particulate particulate samplessamples
ImprovesImproves::–– Bead recoveryBead recovery–– ConsistencyConsistency
Magnetic Magnetic Immunoassay/ConcentrationImmunoassay/Concentration
OverviewOverview
PathatrixPathatrixFor the rapid detection of a range pathogenic and/or spoilage For the rapid detection of a range pathogenic and/or spoilage bacterium in food samples. bacterium in food samples. Antibody coated paramagnetic particles selectively binds and Antibody coated paramagnetic particles selectively binds and purifies the target organism from a comprehensive range of complpurifies the target organism from a comprehensive range of complex ex food matrices. food matrices. Only microbial detection system that can Only microbial detection system that can analyseanalyse the the entire 225ml + entire 225ml + 25g25g sample simultaneously by resample simultaneously by re--circulating the sample through a circulating the sample through a "capture phase" where the antibody coated magnetic beads are "capture phase" where the antibody coated magnetic beads are immobilized. immobilized. By providing heat to the system the organisms can be cultured anBy providing heat to the system the organisms can be cultured and d captured simultaneously, thus increasing the method sensitivity.captured simultaneously, thus increasing the method sensitivity.Use with a variety of detection methodsUse with a variety of detection methods(direct plating onto selective media, COLORTRIX; FLURATRIX (direct plating onto selective media, COLORTRIX; FLURATRIX (fluorescence microscopy); serology; PCR; ELISA; and/or DNA (fluorescence microscopy); serology; PCR; ELISA; and/or DNA probe). probe).
Figure 1: Antibody coated beads capturing Figure 1: Antibody coated beads capturing on surface of capture phaseon surface of capture phase
The beads are added immediately prior to The beads are added immediately prior to rere--circulating the samplecirculating the sample
Figure 2: Capture of Target in FoodFigure 2: Capture of Target in FoodThe sample is reThe sample is re--circulated repeatedly circulated repeatedly across the capture phase with the whole across the capture phase with the whole 250ml sample passing over the phase 250ml sample passing over the phase approximately twice every minute.approximately twice every minute.
Figure 3: Captured target bacteria after Figure 3: Captured target bacteria after washwashWhen the reWhen the re--circulation is complete the circulation is complete the captured bacteria (bound to the antibody captured bacteria (bound to the antibody coated magnetic beads) can be washed coated magnetic beads) can be washed extensively.extensively.
Figure 4: Captured target bacteria are Figure 4: Captured target bacteria are eluted eluted The magnet is removed and the bead / The magnet is removed and the bead / bacteria complexes are washed off the bacteria complexes are washed off the capture phase, collected and concentrated capture phase, collected and concentrated in a wash tube (supplied as part of the in a wash tube (supplied as part of the standard consumable). standard consumable).
Figure 5: Detection Solutions for purified Figure 5: Detection Solutions for purified target organismstarget organisms
ChromogenicChromogenic Agar Agar OverviewOverview
E. Coli O157:H7
ChromogenicChromogenic substratessubstrates
Plating MediumPlating Medium–– SelectiveSelective–– DifferentialDifferential–– SpecificSpecific–– SensitiveSensitive–– Screening for preliminary confirmationScreening for preliminary confirmation
Saves timesSaves timesSaves costsSaves costs
–– Detection/differentiation by utilizing specific enzyme Detection/differentiation by utilizing specific enzyme activitiesactivities
BA BA
After 20-24 hours at 35-37 C
ChromogenicChromogenic mediummedium
EnterobacterEnterobacter sakazakiisakazakiiBacillus Bacillus anthracisanthracis (R & F Laboratories)(R & F Laboratories)ListeriaListeria monocytogenesmonocytogenesBacillus cereus/Bacillus Bacillus cereus/Bacillus thuringiensisthuringiensisEscherichia coliEscherichia coli O157:H7O157:H7SalmonellaSalmonellaYersiniaYersinia pestispestis (out soon(out soon--R & F Laboratories)R & F Laboratories)
PCR OverviewPCR Overview
Qualicon ProductsQualicon ProductsBAXBAX®® SystemSystem
Automated detection of bacteria in foodAutomated detection of bacteria in food
Worldwide adoptions and Worldwide adoptions and certificationscertifications
AOAC International Official MethodAOAC International Official MethodSalmonella #2003.09; L. Salmonella #2003.09; L. MonocytogenesMonocytogenes #2003.12#2003.12
AOACAOAC--RI Performance Tested MethodRI Performance Tested MethodSalmonella #100201; Salmonella #100201; ListeriaListeria monocytogenesmonocytogenes
#070202; E. Coli O157:H7 #010401; Genus #070202; E. Coli O157:H7 #010401; Genus ListeriaListeria #030502#030502
USDAUSDA--FSIS AdoptionFSIS AdoptionSalmonella #MLG 4C.00; Salmonella #MLG 4C.00; ListeriaListeria monocytogenesmonocytogenes
#MLG 8A.00#MLG 8A.00Health Canada CertificationHealth Canada CertificationSalmonella #MFLPSalmonella #MFLP--29; 29; ListeriaListeria monocytogenesmonocytogenes
#MFLP#MFLP--28 E. coli O157:H7 #MFLP28 E. coli O157:H7 #MFLP--30; 30; EnterobacterEnterobacter sakazakiisakazakii #MFLP#MFLP--2727
Genetics based Genetics based detectiondetection
Food or Environmental Food or Environmental Sample Yes
No
Sample
Enrichment Is it there?
High SensitivityHigh SensitivityFind the target organism at very low Find the target organism at very low levelslevels
High SpecificityHigh SpecificityFind Find onlyonly the target organism, even the target organism, even among high levels of competitorsamong high levels of competitors
Available BAXAvailable BAX®® System AssaysSystem Assays
SalmonellaSalmonella
GenusGenus ListeriaListeria
ListeriaListeria monocytogenesmonocytogenes
E. coli E. coli O157:H7O157:H7
EnterobacterEnterobacter sakazakiisakazakii
BAXBAX®® System System ——Time to ResultsTime to ResultsBAXBAX®® SystemSystem Other MethodsOther Methods
SalmonellaSalmonella Next dayNext day Minimum 2 days Minimum 2 days +Confirmation+Confirmation
E. coli E. coli O157:H7O157:H7 Next dayNext day Next day + ConfirmationNext day + Confirmation88--hr enrichment mediahr enrichment media Same daySame day
L. L. monocytogenesmonocytogenes 48 hours48 hours Minimum 2 days + Minimum 2 days + ConfirmationConfirmation
ListeriaListeria Genus Genus 48 hours48 hours Minimum 2 days + Minimum 2 days + ConfirmationConfirmation2424--hr enrichment mediahr enrichment media Next dayNext day
E. E. sakazakiisakazakii * * Next dayNext day Minimum 2 days + Minimum 2 days + ConfirmationConfirmation
PCR made easy with the PCR made easy with the BAXBAX®® systemsystem
Tableted reagents Tableted reagents packaged in PCR tubespackaged in PCR tubes
-- provide for process controlprovide for process control-- eliminate multiple transferseliminate multiple transfers-- reduce operator errorreduce operator error
Simplicity and Ease of Simplicity and Ease of UseUse
Minimal sample handlingMinimal sample handlingNO DNA ISOLATION!NO DNA ISOLATION!PipettingPipetting important user skillimportant user skillAssays allow multichannel pipetting to minimize Assays allow multichannel pipetting to minimize handshands--on timeon timeTools developed to simplify manipulationsTools developed to simplify manipulationsUniform protocols for sample typesUniform protocols for sample typesDefinitive, not presumptive resultsDefinitive, not presumptive resultsHigh throughput High throughput –– 96 tests/batch (96 tests/batch (poolingpooling))Screen for multiple organisms simultaneouslyScreen for multiple organisms simultaneously
Automated Automated cyclercycler/detector/detectorUniversal cycling protocol allows amplification of multiple targUniversal cycling protocol allows amplification of multiple targets in ets in one batchone batchSophisticated algorithms analyze melting curves of fluorescent Sophisticated algorithms analyze melting curves of fluorescent detection detection WindowsWindows--based interface, with Wizards that prompt you through the based interface, with Wizards that prompt you through the protocol.protocol.ColorColor--coded graphic display of yescoded graphic display of yes--oror--no results in less than four no results in less than four hourshours
No
Yes
Future targets for the BAXFuture targets for the BAX®®systemsystem
Yeast and Mold (just out last week)Yeast and Mold (just out last week)–– Shortened enrichment + PCR assay for quicker time Shortened enrichment + PCR assay for quicker time
to resultto result
Campylobacter Campylobacter –– PCR assay to detect PCR assay to detect C. C. jejunijejuni and and C. coli C. coli in poultryin poultry–– Estimated delivery Estimated delivery -- mid 2005mid 2005
VibrioVibrio–– PCR assay to detect regulated strains in seafoodPCR assay to detect regulated strains in seafood–– Estimated delivery Estimated delivery -- late 2005late 2005
ElectrochemiluminescenceElectrochemiluminescenceOverviewOverview
Electrochemiluminescent Label
BV-TAG™ label
Ruthenium (II) tris-bipyridine NHS ester
UNIQUE LABEL CHEMISTRY•Stable non-isotopic label (ruthenium)•Unique light emitting characteristics•Variety of linking chemistries•Size: ~1000 daltons•Solubility: Aqueous and Organic solvents
Electrochemiluminescence Process
Ruthenium and TPA are oxidized at the surface of the electrode when voltage is applied. TPA loses a proton which reduces the ruthenium to an excited state, causing light to be emitted. Ruthenium is not consumed, so it can be oxidized and excited again as long as TPA is present.
Multiple excitation/emission cycles amplifies the light emitted and increase sensitivity.
Emitted light is measured to determine concentration of analytes in sample.
BioVeris TechnologyBioVeris TechnologyTMTM –– Read CycleRead CycleSample In Bead Capture
Photodiode Photodiode
Simple automated read cycle
• Precise• Sensitive• No operator
interpretation
Read Wash
Photodiode Photodiode
M1M PortabilityM1M PortabilityMolded transport caseMolded transport caseAnalyzer can be used inAnalyzer can be used in
the transport casethe transport caseThe instrument is readyThe instrument is ready
to run after <15 min.to run after <15 min.Case Dimensions: 29Case Dimensions: 29”” X 15X 15”” x x 2828””Total power consumptionTotal power consumption
< 75 W< 75 WCase contains sufficient bulkCase contains sufficient bulkreagents to run over 300 testsreagents to run over 300 tests
BioVerisBioVeris Available Assays for FoodAvailable Assays for Food
Staphylococcal Staphylococcal enterotoxinenterotoxin AAStaphylococcal Staphylococcal enterotoxinenterotoxin BBBotulinumBotulinum neurotoxins A, B, E, & Fneurotoxins A, B, E, & FE. coliE. coli O157O157AnthraxAnthraxAnthrax Lethal Factor Enzymatic AssayAnthrax Lethal Factor Enzymatic AssayBotulinumBotulinum neurotoxin A & B Enzymatic Assaysneurotoxin A & B Enzymatic AssaysRicinRicinSalmonellaSalmonellaListeriaListeriaCampylobacterCampylobacter
RealReal--time PCR/Molecular time PCR/Molecular Beacons OverviewBeacons Overview
WARNEX RAPID PATHOGEN WARNEX RAPID PATHOGEN DETECTIONDETECTION
SENTINEL - ABOUT THE SOFTWARE
DETECTION PROCESS
SIMULTANEOUS DNA AMPLIFICATION + RESULT ANALYSIS
NO HANDS-ON TIME
THE DETECTION PROCESS:Step 1 + 2 + 3
Step 1Enrichment
Step 2DNA extraction
Step 3DNA amplification& detection & analysis
18-24 hrs 20 min 2 hrs
FROM SAMPLE
ON-LINE SUPPORT
CERTIFICATE OF ANALYSIS
BACTERIAL GROWTH
DNA EXTRACTION
RESULTS WITHIN 3 HOURS POST ENRICHMENT
ENRICHMENT
REAL-TIME PCR DETECTION
READY TO USE PLATES & FLEXIWELLSCONTAINING ALL REQUIRED REAGENTS
Controls
Salmonella spp.
E. coli O157:H7
E. coli O157
L. monocytogenes
Listeria spp.
Campylobacter spp. In validation
U.S. AOAC RI performance test
WarnexWarnex SUMMARYSUMMARY
o SPEED
o VERSATILITY
o SIMPLICITY
o ACCURACY
o Real-Time PCR
o Integrated platform (microplates and flexiwells)
o Ready to use microplateso Automatic analysis of resultso Automatic printout of CoA
o Double specificity concept from Primers and Molecular Beaconsdecrease false + results
oPrimers select pathogensoMolecular beacons confirm itspresence
Real-time Pathogen Detection Kits OverviewOverview
New Applied BiosystemsTaqMan® Pathogen Detection Kits
Salmonella enterica, E. coli O157:H7, Listeria monocytogenes, Campylobacter jejuniavailable Fall 2005
• ease-of-use:- single, closed tube analysis- one single program for all bacteria- integrated and automated amplification, detection, data collection and analysis
- no post-PCR processing required• increased specificity• faster results:
- save days until TTR• better results:
- Internal Positive Control to help prevent false negatives
Applied Applied BiosystemsBiosystems RealReal--Time PCR Time PCR Food Testing with Food Testing with TaqManTaqMan®® ProbesProbes
PrinciplePrinciple::–– combines the amplification power of PCR combines the amplification power of PCR
(sensitivity) with a hybridization reaction (sensitivity) with a hybridization reaction (specificity) in one tube(specificity) in one tube
Experience:Experience:–– proven 3proven 3rdrd generation technology from the generation technology from the
inventors of realinventors of real--time PCR time PCR AdvantagesAdvantages::–– minimizes handsminimizes hands--on needs compared to on needs compared to
conventional PCRconventional PCR–– no carryno carry--over contamination: single, closed over contamination: single, closed
tube analysistube analysis–– iincreasedncreased specificity: addition of a specificity: addition of a fluorogenicfluorogenic
TaqManTaqMan®® probe between PCR primers confirms probe between PCR primers confirms the presence of the correct PCR product onlythe presence of the correct PCR product only
For more informationFor more information……
Yvonne HaleYvonne [email protected]@doacs.state.fl.us
Grace HallGrace [email protected]@doacs.state.fl.us
FL DACS FL DACS FoodborneFoodborne Pathogen Pathogen Analysis ConferenceAnalysis Conference
July 17July 17--20, 2005 St. Pete Beach, FL20, 2005 St. Pete Beach, FLwww.flworkshop.comwww.flworkshop.com