Molecular Methods for the Detection and Characterization of Foodborne and Environmental Pathogens

TABLE OF CONTENTS

Preface

Acknowledgements

Chapter 1. Microorganisms: Detection and Characterization

1.1. Microbes on Earth

1.2. Microbes, Humans, and Animal Health

1.3. History of Microbial Detection and Characterization

1.4. Basic Microbiology Techniques

1.5. Challenges Associated with Conventional Microbiology Techniques

1.6. Molecular Detection of Microorganisms

1.7. Detection Specificity and Detection Sensitivity

1.8. Commercial Kits for Microbial Detection and Characterization

1.9. Validation of Commercial Kits

1.10. References

Chapter 1—Laboratory Experiments

Lab 1.1 Introduction/Serial Dilutions/Direct Plating

Lab 1.2 Commercial-Based Tests: Pathogen Detection and MPN Enumeration

Lab 1.3 Selective Enrichment, Isolation, and MPN Enumeration of Fecal Coliforms and Salmonella in Environmental and Food Samples

Chapter 2. Microbial DNA Extraction, Purification, and Quantification

2.1. Nucleic Acid Extraction Principles

2.2. References

Chapter 2—Laboratory Experiments

Lab 2.1 Genomic DNA Extraction from Pure Bacterial Culture and Community DNA from Environmental and Food Samples

Lab 2.2 Purification, Quantification, and Purity Check of Extracted DNA

Chapter 3. Introduction to PCR Amplification

3.1. Conventional PCR Amplification of Target Sequences

3.2. PCR Amplification from Food and Environmental Samples

3.3. References

Chapter 3—Laboratory Experiments

Lab 3.1 Polymerase Chain Reaction (PCR) of Pathogen Targets and Agarose Gel Electrophoresis

Chapter 4. DNA Fingerprinting

4.1. Bacterial Characterization Methods

4.2. Rationale for DNA Fingerprinting

4.3. DNA Fingerprinting Methods

4.4. Non-PCR-Based Methods

4.5. Foodborne Disease Monitoring Programs in the United States

4.6. PCR-Based Methods

4.7. References

Chapter 4—Laboratory Experiments

Lab 4.1 DiversiLab on a Chip: Bacterial Fingerprinting using the DiversiLab System

Chapter 5. Microbial RNA Extraction, Purification, and Quantification

5.1. Stabilization of RNA Molecules

5.2. Extraction of RNA Molecules

5.3. Protection of Extracted RNA Molecules against RNA Degradation

5.4. Storage of RNA Molecules

5.5. Verifying RNA Concentration and RNA Purity

5.6. References

Chapter 5—Laboratory Experiments

Lab 5.1 Pathogen Total RNA Preservation, Extraction, and Quantification

Lab 5.2 Total Extracted RNA Quality Analysis using the Bioanalyzer

Chapter 6. Gene Expression using Real-Time PCR Assay

6.1. Overview of Real-Time PCR Assays

6.2. Quantifying Microbial Gene Expression using RT-PCR Amplifications

Chapter 6—Laboratory Experiments

Lab 6.1 cDNA Synthesis from Extracted RNA

Lab 6.2 Gene Expression Analysis of Bacterial (Salmonella) Virulence Genes Measured by Real-time PCR using SYBR Green Fluorescence

Appendix A

Appendix B

Index