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Chapter 47 : Molecular Source Tracking and Molecular Subtyping

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Abstract:

This chapter focuses on subtyping for molecular epidemiology. Properties of methods commonly used for molecular subtyping of foodborne pathogens are discussed in the chapter. Subtyping methods includes plasmid profile analysis, restriction fragment length polymorphism (RFLP) methods and ribotyping. Although the focus of the chapter is on molecular methods, it is important to consider them in the context of earlier phenotypic methods such as serotyping, phage typing, biotyping, and antimicrobial susceptibility typing. Perhaps the most important reason for using molecular methods is that they do not require specialized reagents or expertise and can thus be performed in almost any laboratory with basic molecular biology capabilities. This same ease of performance is also the most obvious drawback to molecular subtyping, as it allows every laboratory to perform and interpret subtyping according to their own criteria, making interlaboratory comparisons more difficult unless laboratories can agree to standardized protocols. Specific applications of ribotyping in general and automated ribotyping using the RiboPrinter are discussed. The application of pulsed-field gel electrophoresis (PFGE) to specific foodborne pathogens is described. Applications of amplified fragment length polymorphism (AFLP) to specific foodborne bacteria are discussed in the chapter. For the past several years, PFGE has also been the gold standard for molecular subtyping of .

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47

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Restriction Fragment Length Polymorphism
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Random Amplified Polymorphic DNA
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Pulsed-Field Gel Electrophoresis
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Figures

Image of Figure 47.1
Figure 47.1

Example of plasmid profile analysis. Each lane contains plasmid DNA extracted from an isolate of ceftriaxone-resistant . In this instance, plasmid DNA was separated by PFGE rather than standard gel electrophoresis for better separation of large plasmids.

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Image of Figure 47.2
Figure 47.2

Automated ribotyping using DuPont RiboPrinter. Output shows appearance of blotted and hybridized gel (above) and software-normalized image (below). Identification and subtype (RiboGroup) are automatically generated.

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Image of Figure 47.3
Figure 47.3

PFGE analysis of seven isolates of serotype Berta. (a) Raw data: lanes 2 through 4 and 6 through 9 contain BlnI digests of serotype Berta genomic DNA; lanes 1, 5, and 10 contain XbaI digests of a molecular-weight-standard strain. (b) Analyzed data: dendrogram showing relatedness of serotype Berta isolates. The dendrogram was produced by using BioNumerics software.

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Image of Figure 47.4a
Figure 47.4a

Example of MLVA of O157:H7 isolates. (Top and center) Chromatograms from automated DNA sequencer showing different fragment sizes at three of four sites. nt, nucleotides. (Bottom) Dendrogram showing relatedness of isolates based on MLVA data. vals, values.

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Image of Figure 47.4b
Figure 47.4b

Example of MLVA of O157:H7 isolates. (Top and center) Chromatograms from automated DNA sequencer showing different fragment sizes at three of four sites. nt, nucleotides. (Bottom) Dendrogram showing relatedness of isolates based on MLVA data. vals, values.

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Image of Figure 47.5
Figure 47.5

Example of output from automated DNA sequencing analysis. (Top) Chromatogram showing unanalyzed data (base calls). (Bottom) Analyzed data (aligned sequences).

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47
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Tables

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Table 47.1

Properties of methods commonly used for molecular subtyping of foodborne pathogens

Citation: Barrett T, Gerner-Smidt P. 2007. Molecular Source Tracking and Molecular Subtyping, p 987-1004. In Doyle M, Beuchat L (ed), Food Microbiology: Fundamentals and Frontiers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815912.ch47

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