NatanGollopElangomathavan

TheVolcaniCenter,AROIsrael

Contents(Microbiologicalquantificationmethods)1.Conventionalmicrobiologicaltestmethods2.Biochemicalcharacterizationmethods3.Immunoassays4.Molecularmethods5.Developmentofrapidmethodsinfoodmicrobiology

Whytodevelopnewdetec1onmethods

• Foodbornepathogensarecausingfoodpoisoning;• Foodbornepathogensarenecessarytobeexaminedinfarms,foodindustriesandmarkets;

• Rapidanddependablemethodsareneededforthedetectionandidentificationofthefoodbornepathogens

• Foodspoilagebacteriacauseeconomicallost

Conven1onalmicrobiologicaltestmethodsAdvantages:reliable,official,cheapfacilitiesDisadvantages:time‐consuming,laborconsumingetc

Examples:Platecountmethodsandthemost‐probable‐number(MPN)methods

Rapiddetec1onmethodsinMicrobiology Types:• Biochemicalcharacterizationmethods;• Immunoassays;• Molecularmethods

Advantages:real‐timedetection,labor‐saving,easyoperation

Disadvantages:non‐official(mostly),highlyskilledlabor,expansive

Biochemicalcharacteriza1onmethods Detectionsystemsbasedonautomaticanalysisofcarbonutilizationandotherbio‐reactions

 Detectionsystemsbasedonwholecellularfattyacidanalysis

Systemsbasedoncarbonu1liza1onandotherbio‐reac1ons Kitsbasedonbio‐reactions　　

  Examples:APISystem；EnterotubeⅡ；Micro‐IDR；MinitekTM；CrystalTMIdentificationSystem:RapIDOneSystem:RapIDTMANAⅡSystem

 Characterizationsystemsbasedoncarbonutilizationorsensitivitiesofpathogenstoantibiotics

  Examples： BiologTMATBRIdentification:API:Vitek　

ExampleIforbio‐reac1onsystems

 API20ERSystem Organisms:G‐bacteria Typesofbio‐reactions:23 Timefortests:18‐24hror38‐48hr Advantages:reliable,portable Limitations:professionalworkersareneeded

Exampleforbio‐reac1onsystems�Biolog  Typeofcarbonsources:95 Biologdatabasefor2000speciesofmicroorganisms,including:

 AerobicG‐:526 AerobicG+:339 Anaerobes:361 Yeasts:26 Filamentusfungi:618

 Accuracy:>95%

SystemsbasedonwholecellularfaCyacidanalysisExample：Sherlock®MicrobialIdentificationSystem

Disadvantage:Highlyexpensiveinstrument,highlyskilledoperators

TheAerobelibrarycontainsover695Environmentalspecies

and430Clinicalspecies.

TheAnaerobelibrarycontains725species.

Thislibrarycontains190Yeastspecies.

Immunoassays TECRAsystem:basedonEnzyme‐linkedimmunosorbentassay(ELISA)

 VIDASsystem:basedonEnzyme‐linkedFluorescentimmuno‐Assay(ELFA)

 Transia Biocontrol1‐2Test

MolecularmethodsDNAbandingpattern‐basedmethods　MultiplexPCR　Real‐timePCR　Restrictionfragmentlengthpolymorphis(RFLP)　Randomly‐amplifiedpolymorphicDNA(RAPD)Pulsed‐fieldgelelectrophoresis(PFGE)　Ribotyping(RT)　DNAsequence‐basedmethods　rRNAsequencetypingMultilocussequencetyping(MLST)!DNA‐DNAmicroarray

Ribotyping(RT)system

Pulsed‐fieldgelelectrophoresis(PFGE)

Pulsed‐fieldGelElectrophoresis(PFGE)

Mul1locussequencetyping(MLST)

Mul1locussequencetyping(MLST)

DNAsequence‐basedmethods MultiplexPCR RealtimePCR DNAdetectionchips

  Foodbornepathogensfocused:—Salmonella—Vibrioparahaemolyticus—Staphylococcusaureus—Listeriamonocytogenes—Enterobactercea—E.coli—Spoilagebacteria(pseudomonas)

Mul$plexPCR  Twosetsofprimers Identificationbasedontwoormoregenes Confidentlyinidentification InteractionadInterferencebetweenthetwosetsofprimers

 Onlyonebacteriaperreaction

Real$mePCR  Highsensitivity Fast Onlyonegene Onlyonebacteria Expensive(instrumentandkits)

BioteconGmbH

Q–BioanalytivcGmbH

Real$mePCR TaqMan Beacon Sybergreen

Real$mePCR

DNA‐DNAMicroarray

Strategy—DNAamplification+DNAchipsBacteria—morethan10speciesVeryHighAccuracyKeypoint—numberoftargetgenes

Prove‐it™Bacteria

www.chill-on.com MicroArray strategy

Labeling Shearing

Sampling

Detec$on

DNAisola$on

PCR+labeling

Hybridiza$on

Mul$plexPCR+Labeling

Wholegenomeamplifica$on

Labeling

Wholegenomeamplification

Directhybridization

PCR MultiplexPCR

Directhybridization–Simple,straightforward,lowsensitivity.PCR–Highsensitivity,identificationrelayononegeneonly.ManybacteriacanbedetectinonereactionMultiplexPCR‐Highsensitivity,identificationrelaytwo2to3genesonly.Manybacteriacanbedetectinonereaction,complexity.Wholegenomeamplification–Simple,veryhighsensitivity,,identificationrelaymayrelayonseveralgenesnolimitation,.Manybacteriavirusesandmoldscanbedetectinonesinglereaction

OligoMicroArray GenomicDNAextraction

DNAamplification(labeling)

Designtheoligonucleotidesprobes

Printingthemicroarray

Hybridization

Detection

identification

www.chill‐on.com

MicroArrayforsixbacteria

Microarrayof16SribosomalRNAgene

PCL–PositivecontrolGPB–Gram+BacteriaGNB‐Gram–BacteriaPSU–PseudomonasESC–E.coliSAL–SalmonellaSTP–StaphylococcusauerusVIB–VibriocholeraeLis–Listeriamonocytoge

16SrRNAMicroArray

www.chill‐on.comFishhomogenatesampleprepara1on

Filterwith5µMporesizepaper(21mes) Filterwith0.45µMporesizepaper

Inculca1ngsixpathogenicandspoilagebacteria(102CFU/ml)

(ControlnoBacteria)

Propidiummonoazide(PMA) NOPMAControl

Wholegenomeamplifica1onandDNAlabeling

DNA‐DNAhybridiza1onagainst16SandgyrBandUniquegenesbased

probes

Propidiummonoazide(PMA)

DNAIsola1onandcleaning

Wholegenomeamplifica1onandDNA

labeling Wholegenome

amplifica1onandDNAlabeling

www.chill‐on.com

WholeGenomeAmplification

1. DNAMarker 2. ControlDNA5ngsuppliedwith–WGAkit3. Fishhomogenate+E.coliWGA(20CFU/ml)4. Fishhomogenate+StaphylococcusWGA(10CFU/ml)

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www.chill‐on.com16SrRNAPCRbased(678F+888R)

confirmationofWGAproduct

1.  DNAMarker2.  Fishhomogenate+E.coli–WGA3.Fishhomogenate+Staphylococcus–WGA4.PureE.coliDNA–5.PureStaphylococcusDNA

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www.chill‐on.com

PCL

PCL

GPB

PSU

S

ESC

S

LIS

S

SAL

S

STA

S

VIB

S

GNB

PCL

+

GYR

G2

PSU

G1

ESC

G1

LIS

G2

SAL

G1

STA

G2

VIB

G1

GYR

G1

PCL

PB

ureC

PSU

algT

ESC

rfbE

LIS

rfA

SAL

ssaT

STA

sarZ

VIB

trh

PCL

Staphylococcus

E.Coli

FishSample

Salmonella Pseudomonas

www.chill‐on.com16SrRNAPCRbased(678F+888R)

confirmationofWGAproduct

1.  DNAMarker2.  Fishhomogenate+E.coli–WGA3.Fishhomogenate+Staphylococcus–WGA4.PureE.coliDNA–5.PureStaphylococcusDNA

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www.chill‐on.com

WholeGenomeAmplification

1. DNAMarker 2. ControlDNA5ngsuppliedwith–WGAkit3. Fishhomogenate+E.coliWGA(20CFU/ml)4. Fishhomogenate+StaphylococcusWGA(10CFU/ml)

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HOW LONG IT’S TAKE?! www.chill‐on.com

Bacterialidentification 0 1 6 11

Hours

Sampling WGA Hybridization 12

Scanning