Post on 07-Jul-2020
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