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Chapter 33 : Heterogeneity and Subtyping

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Abstract:

The high level of genomic diversity among isolates of infecting different individuals is widely documented and has provided the primary approach rather than the phenotyping for strain discrimination of the past decade. The purposes of this chapter are: to demonstrate the extent of genetic diversity observed in and to illustrate the various approaches applied to investigate strain heterogeneity at the intragenic level (microdiversity) and throughout the genome (macrodiversity) as well as extragenomic or mobile elements such as plasmids and insertion sequences; to review genetic mechanisms causing diversity, and their possible effects on the discriminatory power and stability of typing schemes; to assess the potential of these approaches for strain typing in terms of discriminatory power, type ability, reproducibility, and general practical applicability; to identify aspects for future development. Several studies assessed the sequence heterogeneity of , and considerable variation was found by restriction fragment length polymorphism (RFLP). The major and minor flagellins are encoded by and , respectively. To study the population structure of , sequences of and have been analyzed for different strains such that the sequences have revealed extensive heterogeneity. The majority of mutations are synonymous due to the selective pressure. Therefore, genotyping methods monitoring only DNA base substitutions may lead to overestimation of the extent of diversity. Genomic diversity within is now well established and a plethora of molecular typing techniques is available, many of which have been applied successfully to distinguish between isolates.

Citation: Owen R, Taylor D, Wang G, van Doorn L. 2001. Heterogeneity and Subtyping, p 363-378. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch33

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Restriction Fragment Length Polymorphism
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Random Amplified Polymorphic DNA
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DNA Synthesis
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Genetic Recombination
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Figures

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Figure 1

Agarose gel-based DNA fingerprints used in genotyping strains of H. pylori. Panel A shows examples of PFGE patterns of Noil digests for various isolates including strain 60190 (lane 3, from left), NCTC11637 (lane 9), and NCTC11638 (lane 10). Panel B shows examples of AFLP profiles of isolates from six patients before and after antibiotic treatment. The first two lanes in each set are pretreatment samples and the second two lanes are posttreatment (reprinted from reference 47 with permission). Panel C shows HaeIII restriction digest patterns of a 3.1-kb PCR amplicon from vacA of various isolates.

Citation: Owen R, Taylor D, Wang G, van Doorn L. 2001. Heterogeneity and Subtyping, p 363-378. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch33
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Image of Figure 2
Figure 2

Genotyping of isolates using reverse hybridization. Panel A shows the principle of the reverse hybridization line probe assay (LiPA). Specific oligonucleotide probes are tailed and immobilized onto membrane strips in parallel lines. Biotinylated PCR products are denatured and hybridized to the probes at highly stringent conditions. Hybrids are detected by a streptavidin-alkaline phosphatase conjugate and substrate, resulting in a purple precipitate. Panel B shows the LiPA for detection of specific mutations in the 23S rDNA, which are associated with macrolide resistance of Panel C shows the virulence LiPA, permitting simultaneous typing of virulence-associated and genes in a single reverse hybridization step after multiplex PCR.

Citation: Owen R, Taylor D, Wang G, van Doorn L. 2001. Heterogeneity and Subtyping, p 363-378. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch33
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Tables

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Table 1

Guidelines to different genotyping methods and their general applications

Citation: Owen R, Taylor D, Wang G, van Doorn L. 2001. Heterogeneity and Subtyping, p 363-378. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch33

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