ABSTRACT
One of the most challenging issues in food safety is the detection of foodborne pathogens. Since the infectious dose of many pathogens is low, the sensitivity of the diagnostic tool becomes important. Traditionally, the detection and analysis of foodborne pathogens have relied on light or electron microscopy and culture methods. Conventional techniques such as microscopy lack sensitivity, are labor intensive, and require well-trained microscopists for accurate identication and interpretation, particularly for pathogens that are morphologically similar or very small in size or present in very low numbers.1-4
2.1 Introduction .................................................................................................................................... 21 2.2 Molecular Detection Methods........................................................................................................ 22
2.2.1 Polymerase Chain Reaction ............................................................................................... 22 2.2.2 Quantitative PCR .............................................................................................................. 22 2.2.3 Loop-Mediated Isothermal Amplication and Other Isothermal Amplication Methods ....23 2.2.4 Microuidic Chips ............................................................................................................. 24 2.2.5 Microarray Detection ........................................................................................................ 25
2.3 Molecular Typing and Population Genetics ................................................................................... 25 2.3.1 Multilocus Sequence Typing and Mutation Scanning Typing Methods ........................... 25 2.3.2 Population Genetics ........................................................................................................... 26
2.4 Phylogenetic Analysis .................................................................................................................... 27 2.4.1 Distance ............................................................................................................................. 27 2.4.2 Parsimony .......................................................................................................................... 27 2.4.3 Likelihood Methods .......................................................................................................... 28 2.4.4 Rooting Trees .................................................................................................................... 28 2.4.5 Statistical Support for Trees .............................................................................................. 28
2.5 Next-Generation Sequencing ......................................................................................................... 29 2.5.1 Pyrosequencing ................................................................................................................. 29 2.5.2 SOLiD (Sequencing by Oligonucleotide Ligation and Detection) .................................... 31 2.5.3 Illumina (Sequencing by Synthesis) .................................................................................. 31 2.5.4 Ion Torrent Personal Genome Machine............................................................................. 31 2.5.5 Single-Molecule Real-Time and Nanopore Sequencing ................................................... 32
2.6 Comparative Genomics .................................................................................................................. 32 2.7 Genetic Manipulation ..................................................................................................................... 33 2.8 Conclusion ...................................................................................................................................... 34 Acknowledgment ..................................................................................................................................... 34 References ................................................................................................................................................ 34
Special stains are required for the diagnosis of some pathogens. In addition, the diagnostic skills of microscopists can vary greatly from laboratory to laboratory resulting in some infections being misdiagnosed or missed completely.2,4,5 Furthermore, light microscopy can have a low sensitivity of detection, which is particularly relevant, considering that, for some pathogens, just a few individuals within a sample may represent an infectious dose.6,7
In vitro cultivation methods are frequently limited in sensitivity, specicity, or both.4,5,8 Many microorganisms still cannot be cultured, and the isolation of others requires special media and laborious procedures that cannot be routinely performed in small laboratories.2,8 Culture methods are also very expensive and time consuming, and in many cases, several days are required before pathogens can be detected.9,10 In addition, many organisms require strictly controlled conditions during transport of specimens, which also adds to the expense.
