Natan Gollop Elango mathavan The Volcani Center, ARO...
Transcript of Natan Gollop Elango mathavan The Volcani Center, ARO...
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NatanGollopElangomathavan
TheVolcaniCenter,AROIsrael
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Contents(Microbiologicalquantificationmethods)1.Conventionalmicrobiologicaltestmethods2.Biochemicalcharacterizationmethods3.Immunoassays4.Molecularmethods5.Developmentofrapidmethodsinfoodmicrobiology
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Whytodevelopnewdetec1onmethods
• Foodbornepathogensarecausingfoodpoisoning;• Foodbornepathogensarenecessarytobeexaminedinfarms,foodindustriesandmarkets;
• Rapidanddependablemethodsareneededforthedetectionandidentificationofthefoodbornepathogens
• Foodspoilagebacteriacauseeconomicallost
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Conven1onalmicrobiologicaltestmethodsAdvantages:reliable,official,cheapfacilitiesDisadvantages:time‐consuming,laborconsumingetc
Examples:Platecountmethodsandthemost‐probable‐number(MPN)methods
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Rapiddetec1onmethodsinMicrobiology Types:• Biochemicalcharacterizationmethods;• Immunoassays;• Molecularmethods
Advantages:real‐timedetection,labor‐saving,easyoperation
Disadvantages:non‐official(mostly),highlyskilledlabor,expansive
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Biochemicalcharacteriza1onmethods Detectionsystemsbasedonautomaticanalysisofcarbonutilizationandotherbio‐reactions
Detectionsystemsbasedonwholecellularfattyacidanalysis
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Systemsbasedoncarbonu1liza1onandotherbio‐reac1ons Kitsbasedonbio‐reactions
Examples:APISystem;EnterotubeⅡ;Micro‐IDR;MinitekTM;CrystalTMIdentificationSystem:RapIDOneSystem:RapIDTMANAⅡSystem
Characterizationsystemsbasedoncarbonutilizationorsensitivitiesofpathogenstoantibiotics
Examples: BiologTMATBRIdentification:API:Vitek
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ExampleIforbio‐reac1onsystems
API20ERSystem Organisms:G‐bacteria Typesofbio‐reactions:23 Timefortests:18‐24hror38‐48hr Advantages:reliable,portable Limitations:professionalworkersareneeded
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Exampleforbio‐reac1onsystems�Biolog Typeofcarbonsources:95 Biologdatabasefor2000speciesofmicroorganisms,including:
AerobicG‐:526 AerobicG+:339 Anaerobes:361 Yeasts:26 Filamentusfungi:618
Accuracy:>95%
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SystemsbasedonwholecellularfaCyacidanalysisExample:Sherlock®MicrobialIdentificationSystem
Disadvantage:Highlyexpensiveinstrument,highlyskilledoperators
TheAerobelibrarycontainsover695Environmentalspecies
and430Clinicalspecies.
TheAnaerobelibrarycontains725species.
Thislibrarycontains190Yeastspecies.
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Immunoassays TECRAsystem:basedonEnzyme‐linkedimmunosorbentassay(ELISA)
VIDASsystem:basedonEnzyme‐linkedFluorescentimmuno‐Assay(ELFA)
Transia Biocontrol1‐2Test
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MolecularmethodsDNAbandingpattern‐basedmethods MultiplexPCR Real‐timePCR Restrictionfragmentlengthpolymorphis(RFLP) Randomly‐amplifiedpolymorphicDNA(RAPD)Pulsed‐fieldgelelectrophoresis(PFGE) Ribotyping(RT) DNAsequence‐basedmethods rRNAsequencetypingMultilocussequencetyping(MLST)!DNA‐DNAmicroarray
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Ribotyping(RT)system
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Pulsed‐fieldgelelectrophoresis(PFGE)
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Pulsed‐fieldGelElectrophoresis(PFGE)
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Mul1locussequencetyping(MLST)
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Mul1locussequencetyping(MLST)
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DNAsequence‐basedmethods MultiplexPCR RealtimePCR DNAdetectionchips
Foodbornepathogensfocused:—Salmonella—Vibrioparahaemolyticus—Staphylococcusaureus—Listeriamonocytogenes—Enterobactercea—E.coli—Spoilagebacteria(pseudomonas)
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Mul$plexPCR Twosetsofprimers Identificationbasedontwoormoregenes Confidentlyinidentification InteractionadInterferencebetweenthetwosetsofprimers
Onlyonebacteriaperreaction
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Real$mePCR Highsensitivity Fast Onlyonegene Onlyonebacteria Expensive(instrumentandkits)
BioteconGmbH
Q–BioanalytivcGmbH
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Real$mePCR TaqMan Beacon Sybergreen
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Real$mePCR
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DNA‐DNAMicroarray
Strategy—DNAamplification+DNAchipsBacteria—morethan10speciesVeryHighAccuracyKeypoint—numberoftargetgenes
Prove‐it™Bacteria
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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
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OligoMicroArray GenomicDNAextraction
DNAamplification(labeling)
Designtheoligonucleotidesprobes
Printingthemicroarray
Hybridization
Detection
identification
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www.chill‐on.com
MicroArrayforsixbacteria
Microarrayof16SribosomalRNAgene
PCL–PositivecontrolGPB–Gram+BacteriaGNB‐Gram–BacteriaPSU–PseudomonasESC–E.coliSAL–SalmonellaSTP–StaphylococcusauerusVIB–VibriocholeraeLis–Listeriamonocytoge
16SrRNAMicroArray
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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
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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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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
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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
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