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 Hale850850--414414--04080408haley1@doacs.state.fl.ushaley1@doacs.state.fl.us

Grace HallGrace Hall850850--414414--04120412hallg@doacs.state.fl.ushallg@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