2007. 9. 13. Food Microbiology Lab. Korea University

2007. 9. 13.2007. 9. 13.

Food Microbiology Lab. Korea UniversityFood Microbiology Lab. Korea University

Park Jwa-HaengPark Jwa-Haeng

Current and emerging molecular diagnostic

technologies applicable to bacterial food safety

IntroductionIntroduction

Currently used FM testingsCurrently used FM testings

Traditional methodsTraditional methods

The mainstay of FM testingThe mainstay of FM testing

Involve the isolation and enumeration or multipleInvolve the isolation and enumeration or multiple

enrichment and culturing steps enrichment and culturing steps

Take about 3-7daysTake about 3-7days

Immuno-assaysImmuno-assays

Simple, rapid and automatable Simple, rapid and automatable

Enzyme-Linked Immuno-sorbent Assays(ELISA)Enzyme-Linked Immuno-sorbent Assays(ELISA)

Latex agglutination assaysLatex agglutination assays


Nucleic acid-based diagnostic(NAD) assaysNucleic acid-based diagnostic(NAD) assays

Have been successfully applied for the detectionHave been successfully applied for the detection

and identification of food borne pathogenes in aand identification of food borne pathogenes in a

wide range of food types during last decadewide range of food types during last decade

Sensitive and specific detection of single or mul-Sensitive and specific detection of single or mul-

tiful pathogenes in foods tiful pathogenes in foods

This reviewThis review

Molecular technologies for detection of common Molecular technologies for detection of common

bacterial food borne pathogenes concretlybacterial food borne pathogenes concretly


NADs for food NADs for food borne pathogensborne pathogens

General characteristicsGeneral characteristics

Faster turnaround time and improved sensitivity and Faster turnaround time and improved sensitivity and specificity compared to conventional technologiesspecificity compared to conventional technologies

Available in a variety of formats ranging from simple Available in a variety of formats ranging from simple nucleic acid probe hybridization systems to tests in-nucleic acid probe hybridization systems to tests in- corporating amplification of a specific genomic targetcorporating amplification of a specific genomic target

Recently, real-time Recently, real-time in-vitro in-vitro amplification system, bio-amplification system, biosensors, and microarray based platformssensors, and microarray based platforms

Nucleic Acid DiagnosticsNucleic Acid Diagnostics

Molecular targetsMolecular targets

The basis of any NAD assay is a specific nucleic acidThe basis of any NAD assay is a specific nucleic acid target sequence, unique to each speciestarget sequence, unique to each species

To allow for differentiation of the pathogen at bothTo allow for differentiation of the pathogen at both genus and species levels, genus and species levels,

A candidate NAD target should be present in theA candidate NAD target should be present in the cell cell at relatively high copy number at relatively high copy number while beingwhile being sufficiently sufficiently heterologousheterologous at the sequence level at the sequence level


Popularly used target genes Popularly used target genes

Genomic DNA, multicopy rRNAGenomic DNA, multicopy rRNA

genes encoding toxins or virulence factors genes encoding toxins or virulence factors

and genes involved in cellular metabolismand genes involved in cellular metabolism



RNA compared to DNA as target geneRNA compared to DNA as target gene

A labile molecularA labile molecular that is quickly and easily degr- that is quickly and easily degr- aded once the organism is killedaded once the organism is killed

Handling RNA is more difficult than DNAHandling RNA is more difficult than DNA

The advantage of enabling viable organisms to The advantage of enabling viable organisms to be distinguished from nonviable organismsbe distinguished from nonviable organisms


Direct DNA probe based NADsDirect DNA probe based NADs Applied for confirmationApplied for confirmation of the identity of organismsof the identity of organisms following culture based isolation of the foodborne pa-following culture based isolation of the foodborne pathogen of concernthogen of concern

Not require sophisticated equipment Not require sophisticated equipment Simple to perform Simple to perform 101044-10-105 5 bacterial cells of detection limitbacterial cells of detection limit

Commercial NAD assays based on direct nucleic acidCommercial NAD assays based on direct nucleic acid probe technologyprobe technology

AccuprobeAccuprobeRR (GenProbe, CA, USA) for (GenProbe, CA, USA) for Campylobacter Campylobacter spp and spp and LL. monocytogenes. monocytogenes Gene-TrakGene-TrakR R (Neogen, MI, USA) for (Neogen, MI, USA) for E. coliE. coli, , SalmonellaSalmonella and and ListeriaListeria spp spp


NADs based on NADs based on in vitro in vitro amplification amplification technologiestechnologies

General scope of General scope of Polymerase chain reaction(PCR)Polymerase chain reaction(PCR)

Most popular platform applied in NAD assays forMost popular platform applied in NAD assays for food borne pathogensfood borne pathogens

Followed by gel electrophoresis, Southern blot Followed by gel electrophoresis, Southern blot hybridization or a detecting method using spe-hybridization or a detecting method using specific nucleic acid probescific nucleic acid probes

For fluorometric or colorimetric detection of PCR For fluorometric or colorimetric detection of PCR products, labelled probes in membrane and products, labelled probes in membrane and microwell are also usedmicrowell are also used


Food Microorganisms detected by Recently Food Microorganisms detected by Recently developed developed PCR based assays PCR based assays

Campylobacter Campylobacter sppspp - target genes : - target genes : fla A, cadF, ceuE, cdtfla A, cadF, ceuE, cdt and 16S rRNA and 16S rRNA

Listeria and L.monocytogenesListeria and L.monocytogenes - target genes : - target genes : hly, hlyA and prfA hly, hlyA and prfA

Salmonella Salmonella spp and spp and Salmonella typhimuriumSalmonella typhimurium - target genes : - target genes : ogdH, invAogdH, invA

E.coli O174 , O177 and O157:H7E.coli O174 , O177 and O157:H7 - target genes : - target genes : wzx, wzy, stx, vt1, vt2, eaeA, hlyAwzx, wzy, stx, vt1, vt2, eaeA, hlyA


Multiplex PCR assays Multiplex PCR assays for simultaneous for simultaneous detectiondetection of two or more FMOs of two or more FMOs

The simultaneous detection of The simultaneous detection of Salmonella Salmonella sppspp., L. monocytogenes ., L. monocytogenes andand E. coli E. coli 0157:H7 0157:H7 in a total assay time of 30hin a total assay time of 30h The simultaneous detection of The simultaneous detection of E. coli 0157:H7, Salmonella E. coli 0157:H7, Salmonella and and Shigella Shigella


Real - time Real - time in vitro in vitro amplificationamplification technologiestechnologies

General scope of General scope of Real-time PCR Real-time PCR

Refers to the collection of technologies and Refers to the collection of technologies and chemistries that chemistries that monitor monitor the accumulation of the accumulation of PCR product PCR product in a reaction while it is taking placein a reaction while it is taking place compared to endpoint detection of the PCR pro-compared to endpoint detection of the PCR product in conventional PCRduct in conventional PCR

Provide Provide sensitive, quantitative detection of PCR sensitive, quantitative detection of PCR products in a fast turnaround time products in a fast turnaround time in a closedin a closed tube format, thereby significantly reducing thetube format, thereby significantly reducing the risk of contaminationrisk of contamination


A number of different fluorescent probes A number of different fluorescent probes have have been employed in real-time PCR assays include-been employed in real-time PCR assays include- ing Sing SYBR greenYBR greenⅠⅠ, T, TaqMan(5’exonuclease), flu-aqMan(5’exonuclease), fluorescent resonance energy transfer(FRET), mol-orescent resonance energy transfer(FRET), molecular beacons and scorpion probes ecular beacons and scorpion probes


Food Microorganisms detected by Recently Food Microorganisms detected by Recently developed developed real-time PCR based assays real-time PCR based assays

SYBR green SYBR green real –time PCR assaysreal –time PCR assays Salmonella, CampylobacterSalmonella, Campylobacter spp. spp.

TaqManTaqMan real -time PCR assays real -time PCR assays SalmonellaSalmonella(I(InvnvA, A, sefsefA), A), L.monocytogenesL.monocytogenes((hlyhlyA)A)

FRET hybridization probe real –time PCR assaysFRET hybridization probe real –time PCR assays Salmonella, CampylobacterSalmonella, Campylobacter spp. spp.


Multiplex real-time PCR assays for Multiplex real-time PCR assays for simultane-simultaneous detectionous detection of two or more FMOs of two or more FMOs

The simultaneous detection of The simultaneous detection of E. coli 0157:H7 , L. monocytogenes E. coli 0157:H7 , L. monocytogenes andand Salmonella strains Salmonella strains using SYBR green dyeusing SYBR green dye

The simultaneous detection of The simultaneous detection of L. monocytogenes L. monocytogenes and and L.innocuaL.innocua using TaqMan probeusing TaqMan probe



Limitations and other considerations Limitations and other considerations forfor in vitro in vitro amplification NAD testsamplification NAD tests

Despite their demonstration as rapid, sensitiveDespite their demonstration as rapid, sensitive and specific detection method, and specific detection method, in-vitroin-vitro amplifica- amplifica- tion NAD assays tion NAD assays still await general acceptancestill await general acceptance and official approvalsand official approvals as standard methods as standard methods because of the lack of universal validation and because of the lack of universal validation and standardizationstandardization

The generation of false-positive results The generation of false-positive results can becan be originated from dead organisms’ presence inoriginated from dead organisms’ presence in the food samplethe food sample

False-negative results False-negative results can occur because of de-can occur because of de- gradation of target nucleic acid sequences and gradation of target nucleic acid sequences and the presence of substances inhibiting the PCRthe presence of substances inhibiting the PCR


Calling for Calling for an international initiatives to focus onan international initiatives to focus on the development the development of internal and external qualityof internal and external quality assurance programs, suitable sample processingassurance programs, suitable sample processing guidelines and establishment of proficiency ringguidelines and establishment of proficiency ring trialstrials The problem of false positive results can be circu-The problem of false positive results can be circu- mvented by mvented by applying a culture enrichment step applying a culture enrichment step prior to PCR analysisprior to PCR analysis

The problem of false negative results can be over-The problem of false negative results can be over- comed by comed by filtration, centrifugation, use of deterge-filtration, centrifugation, use of deterge- nt and organic solvent treatments, enzyme treat-nt and organic solvent treatments, enzyme treat- ment and sample dilutionment and sample dilution before PCR before PCR


Emerging NAD technologies Emerging NAD technologies for foodborne pathogen for foodborne pathogen

detectiondetection

BiosensorsBiosensors

Definition : a group of devices and technologies thatDefinition : a group of devices and technologies that use a biologically derived material immobilized onuse a biologically derived material immobilized on a detection platforma detection platform to measure the presence of one to measure the presence of one or more analytesor more analytes

Two kinds of Nucleic acid based biosensorsTwo kinds of Nucleic acid based biosensors

Quartz crystal microbalances(QCM) in combinat-Quartz crystal microbalances(QCM) in combinat- ion with PCR of the ion with PCR of the laclac gene has been used to det- gene has been used to detect 1-10 ect 1-10 E. coli E. coli cells from 100ml of watercells from 100ml of water

Optical based biosensors use surface plasmon Optical based biosensors use surface plasmon resonance(SPR) to monitor bio-molecular interact-resonance(SPR) to monitor bio-molecular interact- ions on a surface in real timeions on a surface in real time

Emerging NAD Emerging NAD technologiestechnologies

Novel biosensors with advanced visualization and Novel biosensors with advanced visualization and signal amplification technologies have created thesignal amplification technologies have created the possibility of monitoring single molecular interacti-possibility of monitoring single molecular interacti- onon in real time in real time

The next generation of biosensors will The next generation of biosensors will have applic-have applic- action in all sectors of the molecular diagnosticsaction in all sectors of the molecular diagnostics m- m- arketarket


MicroarraysMicroarrays

Definition : consist of large numbers of probes(eitherDefinition : consist of large numbers of probes(either oligonucleotides or cDNAs) immobilized on a solidoligonucleotides or cDNAs) immobilized on a solid surface such as specially treated glass surface such as specially treated glass

Have been demonstrated for the molecular identifica-Have been demonstrated for the molecular identification of tion of E. coli E. coli O157:H7 and O157:H7 and Campylobacter spp. Campylobacter spp. Fro-Fro- m cultures following PCR amplification of target genesm cultures following PCR amplification of target genes

As microarray technologies matures, these planer ar-As microarray technologies matures, these planer ar- rays are being supplemented by further evolutionsrays are being supplemented by further evolutions including including microbead and suspension microarray formatsmicrobead and suspension microarray formats


Future studiesFuture studies

Current technologies are Current technologies are unable to detect such a unable to detect such a low bacterial load from a food matrix without low bacterial load from a food matrix without either either sample amplification or extensive sample purification sample amplification or extensive sample purification techniques techniques so, these major hurdles have to be overcome beforeso, these major hurdles have to be overcome before biosensors and microarrays will provide ‘real time’biosensors and microarrays will provide ‘real time’ detection of pathogens in food samplesdetection of pathogens in food samples


ConclusionConclusion

The application of nucleic acid diagnostics tests forThe application of nucleic acid diagnostics tests for foodborne pathogen identification foodborne pathogen identification is beginning to m-is beginning to m- ake an impact in this sectorake an impact in this sector

Although costs remain high compared with tradition-Although costs remain high compared with traditional methods, al methods, the reduced turnaround time to results the reduced turnaround time to results isis becoming increasingly important for particular foodbecoming increasingly important for particular food typestypes

On going developments in molecular detection plat-On going developments in molecular detection platforms including biosensors and microarrays togetherforms including biosensors and microarrays together with the increasing awareness of the key criteria for with the increasing awareness of the key criteria for consideration in developing NAD assays consideration in developing NAD assays provide po-provide po- tential for new bioanalytical test methods tential for new bioanalytical test methods that will en-that will en- able multiparameter testing and at line monitoring forable multiparameter testing and at line monitoring for microbial contaminantsmicrobial contaminants

ConclusionConclusion

Thank you !Thank you !

Documents

2007. 9. 13. Food Microbiology Lab. Korea University