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Chapter 12 : Repetitive Sequence-Based PCR Typing of Bacteria and Fungi

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Abstract:

Of the number of molecular typing methods that have been described including multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), DNA sequencing of variable genes, ribotyping, PCR ribotyping, restriction fragment length polymorphism analysis, randomly amplified polymorphic DNA (RAPD) analysis, amplified fragment length polymorphism (AFLP) analysis, and repetitive sequence-based PCR (rep-PCR), this chapter focuses on typing of bacteria and fungi specifically using rep-PCR. The theory of rep-PCR is discussed with a comparison of the workflow for traditional gel-based rep-PCR and an automated commercially available system. It is important that the culture be pure, as rep-PCR primer binding sites are widely distributed among bacterial genera. The repetitive extragenic palindrome (REP) was the first repetitive element described in bacteria. The chromosomal locations of the enterobacterial repetitive intergenic consensus (ERIC) elements differ among species but are conserved throughout the kingdom . In addition to bacteria, various fungi have been associated with nosocomial infections. Filamentous fungi, such as species and the Zygomycetes, also contribute to the growing list of health care-associated (HA) infections. While the debate continues over the most cost-effective approach for reducing HA infections, it is clear that some form of an active surveillance program is a key component of effective infection control strategies. Future applications for automated rep-PCR include extension of the menu of both bacterial and fungal pathogens and expansion of the online databases.

Citation: Frye S, Healy M. 2011. Repetitive Sequence-Based PCR Typing of Bacteria and Fungi, p 199-212. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch12

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Restriction Fragment Length Polymorphism
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FIGURE 1

Diagram comparing the workflow of manual or gel-based rep-PCR to the workflow of automated rep-PCR. Specific changes are noted for each step in the workflow.

Citation: Frye S, Healy M. 2011. Repetitive Sequence-Based PCR Typing of Bacteria and Fungi, p 199-212. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch12
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Image of FIGURE 2
FIGURE 2

Graphic illustrating fragment separation. (a) Manual rep-PCR using agarose gel electrophoresis and detection and analysis with camera or scanned imaging and dendrogram created with separate gel compare software. (b) Automated rep-PCR using microfluidics chip and analysis with the accompanying DiversiLab software.

Citation: Frye S, Healy M. 2011. Repetitive Sequence-Based PCR Typing of Bacteria and Fungi, p 199-212. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch12
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Image of FIGURE 3
FIGURE 3

Graphic of the interpretation tool provided in the DiversiLab software to aid with analysis and interpretation. (A) Steps of analysis indicated by tabs; (B) patterns can be assigned “rep” types to organize samples and libraries; (C) users can easily toggle between virtual gel images and sample graphs; (D and E) pop-up overlays can be visualized to better compare two samples.

Citation: Frye S, Healy M. 2011. Repetitive Sequence-Based PCR Typing of Bacteria and Fungi, p 199-212. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch12
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