Rapid Detection and Characterization of Foodborne Pathogens by Molecular Techniques

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ISBN: 9781420092424 | 1420092421
Cover: Hardcover
Copyright: 10/26/2009

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Written by one of the most prolific and respected researchers in food safety, this volume describes molecular techniques for the detectionand discrimination of major infectious bacteria associated with foods. Each chapter deals with a specific organism and techniques applied to that organism. Particular focus is placed on genes associated with pathogenicity used in the polymerase chain reaction (PCR) including real-time PCR for specific detection of pathogenic bacteria and the inherent limitations of such methodology with certain pathogens. Methods for extracting microorganisms from complex food matrices andDNA purification techniques are also emphasized.

Prefacex	p. iii
The Author	p. xv
Molecular Techniques for Detecting, Quantifying, and Subspecies Typing of Foodborne Pathogenic Bacteria	p. 1
The Polymerase Chain Reaction (PCR)	p. 1
Introduction	p. 1
Requirements for PCR	p. 1
Sample Preparation without Enrichment	p. 3
Lysing of Cells and Isolation of Bacterial DNA	p. 3
PCR for Identification of Pure Cultures	p. 4
Quantitative PCR	p. 4
Use of an Internal Standard for Quantitative PCR	p. 5
Construction of an Internal Standard for Quantitative PCR	p. 6
Construction of a Calibration Curve for Quantitative PCR	p. 7
Real-Time PCR (Rti-PCR)	p. 8
Introduction	p. 8
Advantages of Rti-PCR	p. 8
Mechanisms of Rti-PCR	p. 10
SYBR Green	p. 10
TaqMan™ Probes	p. 11
Fluorescent Resonance Energy Transfer (FRET)	p. 12
Molecular Beacons™	p. 14
Unique Fluorogenic Primers	p. 15
Theory of Quantitative Real-Time PCR (QRti-PCR)	p. 16
Problems and Limitations of QRti-PCR	p. 19
Nested PCR	p. 20
Introduction	p. 20
Loop-Mediated Isothermal Amplification (LAMP) Assay	p. 22
Introduction	p. 22
Pulsed-Field Gel Electrophoresis (PFGE)	p. 23
Introduction	p. 23
Mechanism of PFGE	p. 23
Interpretation of PFGE Banding Patterns	p. 24
Random Amplification of Polymorphic DNA (RAPD)	p. 25
Introduction	p. 25
Mechanism of RAPD	p. 26
Multilocus Sequence Typing (MLST)	p. 27
Introduction	p. 27
Restriction Fragment Length Polymorphism (RFLP) and PCR-RFLP	p. 27
Introduction	p. 27
Amplified Fragment Length Polymorphism (AFLP)	p. 28
Introduction	p. 28
References	p. 28
Escherichia coli 0157.H7	p. 33
Characteristics of the Organism	p. 33
Virulence Factors	p. 33
Hemolysins	p. 33
Intimin	p. 34
Shiga-Like Toxins	p. 34
Locus for Enterocyte Effacement	p. 34
Extracellular Serine Protease	p. 35
Additional Virulence Factors	p. 35
Phenotypic Variation of E. coli 0157:H7	p. 35
Conventional PCR	p. 36
Multiplex PCR	p. 49
PCR Assays Involving Molecular Probes and Real-Time PCR (Rti-PCR)	p. 52
Loop-Mediated Isothermal Amplification (LAMP) of DNA	p. 54
Immunomagnetic Separation and PCR	p. 54
Restriction Fragment Length Polymorphism	p. 55
Subspecies Typing of coli 0157:H7 Isolates	p. 55
References	p. 56
Shigella	p. 63
Characteristics of the Genus	p. 63
Virulence Factors	p. 64
PCR Detection of Shigellas	p. 65
Conventional PCR Assays	p. 65
Microarrays	p. 71
Multiplex PCR	p. 72
Immunocapture PCR	p. 73
Molecular Typing	p. 73
References	p. 75
Salmonella	p. 79
Characteristics of the Genus	p. 79
Molecular Techniques	p. 82
Conventional PCR	p. 82
Real-Time PCR (Rti-PCR)	p. 110
Multiplex PCR (mPCR)	p. 115
Pulsed Field Gel Electrophoresis (PFGE)	p. 122
Subtracted Restriction Fingerprinting (SFP)	p. 127
Random Amplified Polymorphic DNA (RAPD) Analysis	p. 127
References	p. 129
Vibrio vulnificus	p. 139
Characteristics of the Organism	p. 139
Virulence Factors	p. 140
Capsule Production	p. 140
Extracellular Virulence Factors	p. 141
Serum-Iron Availability	p. 141
Enrichment and Isolation Media	p. 142
Alkaline Peptone Salt Broth (APS)	p. 142
Colistin-PoIymyxin-p-Cellobiose (CPC) Agar	p. 142
Thiosulfate-Citrate-Bile-Salts Sucrose (TCBS) Agar	p. 142
Vibrio vulnificus (VV) Agar	p. 143
Sodium Dodecyl Sulfate-Polymyxin-Sucrose (SPS) Agar	p. 143
Cellobiose-Colistin (CC) Agar	p. 143
Vibrio vulnificus Medium (VVM)	p. 143
Identification of V. vulnificus Isolates	p. 143
Biochemical Characteristics	p. 143
Serotyping and Immunological Techniques	p. 144
Typing of V. vulnificus Isolates below the Species Level	p. 145
Biogroups	p. 145
Viable but Nonculturable (VBNC) V. vulnificus	p. 152
Molecular Methods for Detection and Typing	p. 153
Conventional PCR	p. 153
Multiplex PCR	p. 154
Real-Time PCR (Rti-PCR)	p. 155
RAPD	p. 157
Ribotyping	p. 158
16S rRNA Sequencing	p. 159
Oligonucleotide Probe	p. 159
References	p. 161
Vibrio parahaemulyiicus	p. 167
Characteristics of the Organism	p. 167
Clinical Symptoms Due to Infections by V. parahaemulyiicus	p. 168
Ecology of V. parahaemolyticus	p. 168
Phenotypic Characteristics of V. parahaemolyticus	p. 169
General	p. 169
Flagellation	p. 170
Antigenic Properties	p. 171
Hemolysins	p. 172
General	p. 172
Direct Acting Hemolysins	p. 172
H2S Production	p. 179
Urease (Uh) Production	p. 179
Sensitivity of V. parahaemolyticus to Low Temperatures	p. 180
Isolation and Cultivation of V. parahaemolyticus	p. 181
Preservation of V. parahaemolyticus Isolates	p. 182
Bacteriophage for V parahaemolyticus	p. 182
Use of PCR for Detection of V. parahaemolyticus	p. 182
Molecular Typing of V. parahaemolyticus Isolates below the Species Level	p. 188
The 03:K6 Pandemic Clone	p. 188
References	p. 192
Vibrio cholerae	p. 203
Characteristics of the Organism	p. 203
Factors Associated with the Virulence of V. cholerae	p. 204
PCR Detection, Identification, and Characterization of V. cholerae	p. 204
Molecular Typing of V. cholerae Isolates	p. 223
Acquisition of Antibiotic Resistance	p. 227
Biotype Conversion by Phage	p. 227
References	p. 229
Aeromonas hydrophila	p. 235
Characteristics of the Organism	p. 235
PCR Detection	p. 236
Distribution of Toxin Genes	p. 237
Genotyping Using Hemolysin and Aerolysin Genes	p. 241
Restriction Fragment Length Polymorphism (RFLP)	p. 241
References	p. 242
Plesiomonas shigelloides	p. 245
Characteristics of the Organism	p. 245
Nomenclature and Taxonomy	p. 245
Physiological and Biochemical Characteristics	p. 246
Ecological Distribution	p. 246
Toxins and Invasive Factors	p. 248
(ß-Hemolysis	p. 249
Isolation	p. 249
Serology	p. 250
Epidemiology and Outbreaks	p. 252
Application of PCR	p. 252
Conventional PCR	p. 252
Real-Time PCR (Rti-PCR)	p. 255
References	p. 256
Campylobacter Jejuni	p. 261
Characteristics of the Organism	p. 261
Phenotypic Characteristics of Campylobacters	p. 262
Ecological Distribution of Campylobacters	p. 262
Environmental Factors	p. 262
Aquatic Sources of Campylobacters	p. 263
Wildlife as a Potential Reservoir for Infection by C. Jejuni	p. 264
Campylobacters Associated with Farm and Domesticated Animals	p. 266
Vertical versus Horizontal Transmission among Poultry	p. 269
Virulence Factors	p. 269
Toxins	p. 269
Cell Adhesion Factors	p. 275
Lipopolysaccharide (LPS)	p. 276
Capsule Formation	p. 277
Virulence Plasmid'	p. 277
Isolation of Campylobacters from Foods	p. 277
Serotyping of Campylobacters and Immunological Detection	p. 283
Bacteriophage Typing of C. jejuni	p. 284
Molecular Methods of Detecting and Typing Campylobacters	p. 285
Genes Used	p. 285
PCR	p. 285
Randomly Amplified Polymorphic DNA (RAPD) Analysis	p. 305
Pulsed Field Gel Electrophoresis (PFGE)	p. 307
Restriction Fragment Length Polymorphism (RFLP) Analysis	p. 310
Amplified Fragment Length Polymorphism (AFLP)	p. 313
In Situ Colony Hybridization	p. 313
Multilocus Sequence Typing (MLST)	p. 314
Loop-Mediated Isothermal Amplification (LAMP)	p. 315
Nucleic Acid Sequence Based Amplification (NASBA)	p. 315
Restriction Endonuclease Analysis	p. 316
The Viable but Nonculturable (VBNC) State of Campylobacters	p. 316
The Coccoid Form of C. jejuni	p. 319
Immunomagnetic Capture of C. jejuni	p. 319
References	p. 320
Staphylococcus aureus	p. 337
Characteristics of the Organism	p. 337
Molecular Techniques for Detection and Identification of S. aureus	p. 338
PCR Used to Detect and Identify S. aureus	p. 338
Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH) for Detection of 5. aureus	p. 341
Conventional and Real-Time PCR (Rti-PCR) Detection of Enterotoxin Genes	p. 341
Loop-Mediated Isothermal Amplification (LAMP) Assay for se Toxin Genes	p. 348
PCR Detection of the tst Gene in S. aureus Strains	p. 349
Molecular Typing of S. aureus Isolates (RAPD, PFGE, RFLP, PCR-RFLP)	p. 349
Multilocus Sequence Typing (MLST) of S. aureus Isolates	p. 355
PCR-Immuno Assays for Detection of S. aureus Exotoxin Genes	p. 358
Exfoliative Toxin (ET) Producing Strains of 5. aureus	p. 359
PCR Detection of Methicillin-Resistant S. aureus Strains	p. 360
Enterotoxin Production by S. intermedins	p. 362
The Role of S. aureus Bacteriophage (Phage) in Pathogenesis	p. 362
References	p. 401
Listeria monocytogenes	p. 411
Characteristics of the Organism	p. 411
Gene Sequences Used in PCR Assays for L. monocytogenes	p. 412
PCR Identification of L. monocytogenes and Other Members of the Genus Listeria	p. 413
Direct PCR Detection of L. monocytogenes in Foods without Enrichment Cultivation	p. 422
PCR Detection of L. monocytogenes in Foods Following Enrichment Cultivation	p. 424
Nested PCR	p. 427
Multiplex PCR (mPCR)	p. 429
Real-Time PCR (Rti-PCR)	p. 431
Application of Random Amplified Polymorphic DNA (RAPD) Analysis to L. monocytogenes Isolates	p. 432
Application of Pulsed Field Gel Electrophoresis (PFGE) to L. monocytogenes Isolates	p. 441
Comparison of RAPD, PFGE, and Other Molecular Methods for Typing L. monocytogenes Isolates	p. 445
Microarray Technology	p. 448
PCR Determination of Serotype Divisions	p. 448
References	p. 449
Clostridium botulinum	p. 457
Characteristics of the Organism	p. 457
Relationship between Botulism and Seafood	p. 457
Infant Botulism	p. 458
Historical Aspects and Incidence	p. 458
Implication of Honey in Infant Botulism	p. 459
Molecular Techniques Applied to C. botulinum	p. 460
PCR Detection of C. botulinum	p. 460
Molecular Typing of Clostridium botulinum Strains	p. 465
Molecular Methods for Differentiating between Group I and Group II Strains	p. 468
Molecular Methods for Detection of BoNTs	p. 469
Reverse Transcription PCR for Indirect Measurement of BoNT Production	p. 469
Immunopolymerase Chain Reaction Assays for Ultrasensitive BoNT Detection	p. 470
References	p. 481
Clostridium perfringens	p. 487
Characteristics of the Organism	p. 487
Molecular Methods for Detection of C. perfringens	p. 488
PCR	p. 488
Molecular Probes	p. 504
Molecular Typing of Isolates	p. 506
Molecular Organization of the CPE Gene	p. 506
Genotyping	p. 510
Random Amplified Polymorphic DNA (RAPD) Typing	p. 514
Pulsed Field Gel Electrophoresis (PFGE) Typing	p. 514
Amplified Fragment Length Polymorphism (AFLP)	p. 515
Ribotyping	p. 516
Multiple-Locus Variable Number Tandem Repeat Loci (VNTR)	p. 517
References	p. 517
Bacillus cereus	p. 523
Characteristics of the Organism	p. 523
Selective Isolation of B. cereus	p. 524
Selective Agar Media	p. 524
The Emetic Toxin Cereulide	p. 525
Synthesis of Cereulide and Location of the-Responsible Peptide Synthetase	p. 525
Phenotypic Characteristics of Emetic Toxin Producing Strains	p. 525
RAPD Typing of Emetic Strains of B. cereus	p. 525
Enterotoxins of B. cereus	p. 526
Major Enterotoxins of B. cereus	p. 526
Molecular Methods for Detection and Confirmation of B. cereus	p. 526
PCR	p. 526
PCR Detection of Emetic Strains of B. cereus	p. 526
PCR Detection and Confirmation of B. cereus and Members of the B. cereus Group	p. 529
Molecular Typing of B. cereus Isolates	p. 550
Genotyping of B. cereus Isolates	p. 550
Multilocus Sequence Typing (MLST) of B. cereus Isolates	p. 556
RAPD Typing	p. 556
Ribotyping	p. 556
PCR Restriction Fragment Length Polymorphism (PCR-RFLP)	p. 557
Microarrays	p. 557
References	p. 559
Index	p. 565
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Rapid Detection and Characterization of Foodborne Pathogens by Molecular Techniques

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