Transcript Changes in heat tolerance of Escherichia coli 0157:H7

Current and emerging molecular
diagnostic technologies applicable
to bacterial food safety
2007. 9. 13.
Food Microbiology Lab. Korea University
Park Jwa-Haeng
Introduction
Introduction
 Currently used FM testings
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Traditional methods
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The mainstay of FM testing
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Involve the isolation and enumeration or multiple
enrichment and culturing steps
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Take about 3-7days
Immuno-assays
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Simple, rapid and automatable
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Enzyme-Linked Immuno-sorbent Assays(ELISA)
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Latex agglutination assays
Introduction
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Nucleic acid-based diagnostic(NAD) assays
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Have been successfully applied for the detection
and identification of food borne pathogenes in a
wide range of food types during last decade
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Sensitive and specific detection of single or multiful pathogenes in foods
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This review
Molecular technologies for detection of common
bacterial food borne pathogenes concretly
NADs for food borne
pathogens
Nucleic Acid Diagnostics
 General characteristics
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Faster turnaround time and improved sensitivity and
specificity compared to conventional technologies
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Available in a variety of formats ranging from simple
nucleic acid probe hybridization systems to tests incorporating amplification of a specific genomic target
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Recently, real-time in-vitro amplification system, biosensors, and microarray based platforms
Nucleic Acid Diagnostics
 Molecular targets
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The basis of any NAD assay is a specific nucleic acid
target sequence, unique to each species
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To allow for differentiation of the pathogen at both
genus and species levels,
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A candidate NAD target should be present in the
cell at relatively high copy number while being
sufficiently heterologous at the sequence level
Nucleic Acid Diagnostics
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Popularly used target genes
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Genomic DNA, multicopy rRNA
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genes encoding toxins or virulence factors
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and genes involved in cellular metabolism
Nucleic Acid Diagnostics
Nucleic Acid Diagnostics
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RNA compared to DNA as target gene
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A labile molecular that is quickly and easily degraded once the organism is killed
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Handling RNA is more difficult than DNA
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The advantage of enabling viable organisms to
be distinguished from nonviable organisms
Nucleic Acid Diagnostics
 Direct DNA probe based NADs
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Applied for confirmation of the identity of organisms
following culture based isolation of the foodborne pathogen of concern
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Not require sophisticated equipment
 Simple to perform
 104-105 bacterial cells of detection limit
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Commercial NAD assays based on direct nucleic acid
probe technology
 AccuprobeR (GenProbe, CA, USA) for
Campylobacter spp and L. monocytogenes
 Gene-TrakR (Neogen, MI, USA) for
E. coli, Salmonella and Listeria spp
Nucleic Acid Diagnostics
 NADs based on in vitro amplification technologies
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General scope of Polymerase chain reaction(PCR)
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Most popular platform applied in NAD assays for
food borne pathogens
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Followed by gel electrophoresis, Southern blot
hybridization or a detecting method using specific nucleic acid probes
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For fluorometric or colorimetric detection of PCR
products, labelled probes in membrane and
microwell are also used
Nucleic Acid Diagnostics
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Food Microorganisms detected by Recently
developed PCR based assays
 Campylobacter spp
- target genes : fla A, cadF, ceuE, cdt and 16S rRNA
 Listeria and L.monocytogenes
- target genes : hly, hlyA and prfA
 Salmonella spp and Salmonella typhimurium
- target genes : ogdH, invA
 E.coli O174 , O177 and O157:H7
- target genes : wzx, wzy, stx, vt1, vt2, eaeA, hlyA
Nucleic Acid Diagnostics
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Multiplex PCR assays for simultaneous
detection of two or more FMOs
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The simultaneous detection of
Salmonella spp., L. monocytogenes
and E. coli 0157:H7
in a total assay time of 30h
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The simultaneous detection of
E. coli 0157:H7, Salmonella and Shigella
Nucleic Acid Diagnostics
 Real - time in vitro amplification technologies
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General scope of Real-time PCR
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Refers to the collection of technologies and
chemistries that monitor the accumulation of
PCR product in a reaction while it is taking place
compared to endpoint detection of the PCR product in conventional PCR
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Provide sensitive, quantitative detection of PCR
products in a fast turnaround time in a closed
tube format, thereby significantly reducing the
risk of contamination
Nucleic Acid Diagnostics
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A number of different fluorescent probes have
been employed in real-time PCR assays includeing SYBR greenⅠ, TaqMan(5’exonuclease), fluorescent resonance energy transfer(FRET), molecular beacons and scorpion probes
Nucleic Acid Diagnostics
 Food Microorganisms detected by Recently
developed real-time PCR based assays
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SYBR green real –time PCR assays
 Salmonella, Campylobacter spp.
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TaqMan real -time PCR assays
 Salmonella(InvA, sefA), L.monocytogenes(hlyA)
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FRET hybridization probe real –time PCR assays
 Salmonella, Campylobacter spp.
Nucleic Acid Diagnostics
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Multiplex real-time PCR assays for simultaneous detection of two or more FMOs
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The simultaneous detection of
E. coli 0157:H7 , L. monocytogenes
and Salmonella strains
using SYBR green dye
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The simultaneous detection of
L. monocytogenes and L.innocua
using TaqMan probe
Nucleic Acid Diagnostics
Nucleic Acid Diagnostics
 Limitations and other considerations for
in vitro amplification NAD tests
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Despite their demonstration as rapid, sensitive
and specific detection method, in-vitro amplification NAD assays still await general acceptance
and official approvals as standard methods
because of the lack of universal validation and
standardization
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The generation of false-positive results can be
originated from dead organisms’ presence in
the food sample
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False-negative results can occur because of degradation of target nucleic acid sequences and
the presence of substances inhibiting the PCR
Nucleic Acid Diagnostics
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Calling for an international initiatives to focus on
the development of internal and external quality
assurance programs, suitable sample processing
guidelines and establishment of proficiency ring
trials
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The problem of false positive results can be circumvented by applying a culture enrichment step
prior to PCR analysis
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The problem of false negative results can be overcomed by filtration, centrifugation, use of detergent and organic solvent treatments, enzyme treatment and sample dilution before PCR
Emerging NAD technologies
for foodborne pathogen
detection
Emerging NAD
technologies
 Biosensors
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Definition : a group of devices and technologies that
use a biologically derived material immobilized on
a detection platform to measure the presence of one
or more analytes
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Two kinds of Nucleic acid based biosensors
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Quartz crystal microbalances(QCM) in combination with PCR of the lac gene has been used to detect 1-10 E. coli cells from 100ml of water
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Optical based biosensors use surface plasmon
resonance(SPR) to monitor bio-molecular interactions on a surface in real time
Emerging NAD
technologies
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Novel biosensors with advanced visualization and
signal amplification technologies have created the
possibility of monitoring single molecular interaction in real time
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The next generation of biosensors will have applicaction in all sectors of the molecular diagnostics market
Emerging NAD
technologies
 Microarrays
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Definition : consist of large numbers of probes(either
oligonucleotides or cDNAs) immobilized on a solid
surface such as specially treated glass
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Have been demonstrated for the molecular identification of E. coli O157:H7 and Campylobacter spp. From cultures following PCR amplification of target genes
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As microarray technologies matures, these planer arrays are being supplemented by further evolutions
including microbead and suspension microarray formats
Emerging NAD
technologies
 Future studies
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Current technologies are unable to detect such a
low bacterial load from a food matrix without either
sample amplification or extensive sample purification
techniques
so, these major hurdles have to be overcome before
biosensors and microarrays will provide ‘real time’
detection of pathogens in food samples
Conclusion
Conclusion
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The application of nucleic acid diagnostics tests for
foodborne pathogen identification is beginning to make an impact in this sector
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Although costs remain high compared with traditional methods, the reduced turnaround time to results is
becoming increasingly important for particular food
types
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On going developments in molecular detection platforms including biosensors and microarrays together
with the increasing awareness of the key criteria for
consideration in developing NAD assays provide potential for new bioanalytical test methods that will enable multiparameter testing and at line monitoring for
microbial contaminants
Thank you !