ABSTRACT
CONTENTS 9.1 Detection of Food-Borne Pathogens .................................................... 214
9.1.1 Conventional Methods............................................................. 215 9.1.2 Rapid Methods .......................................................................... 215
9.1.2.1 Immunoassays............................................................ 215 9.1.2.2 Nucleic Acid-Based Methods .................................. 215
9.2 Conventional PCR .................................................................................. 216 9.2.1 Detection of PCR Products........................................................ 217
9.3 Real-Time PCR ........................................................................................ 218 9.3.1 Real-Time Detection Chemistries........................................... 218
9.4 Advantages of PCR-Based Methods.................................................... 221 9.5 Concerns with the Use of PCR-Based Methods ................................ 222 9.6 PCR Troubleshooting ............................................................................. 223 9.7 Conventional and Real-Time Multiplex PCR .................................... 224 9.8 Detection and Identification of Microorganisms by the PCR ......... 225
9.8.1 Bacteria ....................................................................................... 225 9.8.2 Yeasts and Molds...................................................................... 229 9.8.3 Viruses ........................................................................................ 229 9.8.4 Parasites...................................................................................... 229
9.9 Commercially Available PCR Kits and Related Materials .............. 230 9.10 Quantitative PCR .................................................................................... 231 9.11 Reverse Transcription PCR ................................................................... 232 9.12 Other Nucleic Acid Amplification Methods ...................................... 233
9.12.1 Ligase Chain Reaction.............................................................. 233 9.12.2 Strand Displacement Amplification ...................................... 233
9.12.3 Nucleic Acid Sequence-Based Amplification....................... 234 9.12.4 Rolling Circle Amplification and Ramification
Amplification............................................................................. 234 9.12.5 Qb Replicase .............................................................................. 235 9.12.6 Loop-Mediated Isothermal Amplification............................ 235 9.12.7 Isothermal and Chimeric Primer-Initiated
Amplification of Nucleic Acids .............................................. 237 9.13 DNA Microarrays ................................................................................... 237 9.14 PCR-Based Techniques for Typing Microorganisms........................ 239
9.14.1 Random-Amplified Polymorphic DNA................................ 240 9.14.2 Amplified Restriction Fragment Length Polymorphism ... 240 9.14.3 PCR-Restriction Fragment Length Polymorphism.............. 240 9.14.4 Rep-PCR ..................................................................................... 241 9.14.5 PCR-Ribotyping ........................................................................ 241 9.14.6 Sau-PCR...................................................................................... 241 9.14.7 Denaturing Gradient Gel Electrophoresis
and Temperature Gradient Gel Electrophoresis.................. 242 9.14.8 PCR-Single-Strand Conformation
Polymorphisms ......................................................................... 242 9.14.9 16S rDNA Sequencing (Homology Search).......................... 243
9.15 Nanotechnology: On-Chip PCR ........................................................... 243 References ........................................................................................................... 244
Public awareness of microorganisms transmitted by food which pose a severe threat to human health has increased dramatically in recent years. Food-borne pathogenic microorganisms include bacterial pathogens, such as Salmonella and Campylobacter spp., Listeria monocytogenes, Escherichia coli O157:H7 and other Shiga toxin-producing E. coli, Yersinia enterocolitica, and Clostridium perfringens, protozoan parasites, including Giardia, Cryptosporidium, and Cyclospora, and enteric viruses including hepatitis A and noroviruses. In a report from the Centers for Disease Control and Prevention, it was estimated that there are approximately 76 million cases of food-borne illnesses each year in the United States with 325,000 hospitalizations, and 5000 deaths.1 The Council for Agricultural Science and Technology Task Force Report estimated that the annual incidence of microbial food-borne disease cases in the United States ranges from 6.5 to 33 million, with as many as 9000 deaths annually.2
Thus, there is a need for rapid, sensitive, and reliable methods for detection of food-borne pathogens and for investigating cases and outbreaks of illness caused by these agents. Furthermore, development of rapid diagnostic systems that have the capability to link sample processing, preparation, and simultaneous detection of multiple pathogens and new threats, including potential biothreat agents, is important for food safety.
