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Chapter 2 : Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques

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Abstract:

This chapter reviews both conventional and molecular biology laboratory techniques that are used to type organisms that cause human infectious diseases, emphasizing those techniques that are applied to conduct epidemiologic investigations. It classifies all laboratory-typing systems into phenotypic and genotypic methods. Most of the conventional laboratory typing methods fall into the category of phenotypic methods, which are based on the detection of phenotypes or characteristics expressed by an organism. Strain-typing methods based on genotypes rely on the analysis of nucleic acid contents and gene sequence polymorphisms (chromosomal DNA, extrachromosomal DNA, and RNA). The chapter presents an overview of the basic principles behind commonly used nonmolecular as well as non-PCR-based molecular biology analytical techniques applied to type pathogens for epidemiologic investigations.

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2

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Restriction Fragment Length Polymorphism
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Figures

Image of Figure 2.1
Figure 2.1

Phage typing of hypothetical bacteria X and Y. Drops of bacteriophage suspensions from a panel of bacteriophages 1 through 6 are placed onto a lawn of test bacterium grown on agar plates. A bacterium susceptible to the particular phage will show a zone of lysis, which is indicated by a dark gray oval. The lysis pattern determines the phage type of the organism. (Illustration by Ariana Reynolds.)

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2
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Image of Figure 2.2
Figure 2.2

Extraction, purification, and electrophoresis of DNA from two bacterial samples (samples 1 and 2). The sample 1 bacterium harbors four plasmids, and the sample 2 bacterium has two plasmids. The bacterial cell wall, outer membrane (in gram-negative bacteria), and inner membrane are removed by the indicated reagents at alkaline pH (see text for what each of these reagents does). The alkaline pH separates double-stranded DNA into single strands, which are soluble in an aqueous solution. When the pH of the solution is decreased to an acidic range (∼pH 4.5), the single-stranded DNA becomes double-stranded, and large pieces of double-stranded DNA (chromosome) precipitate, while the plasmid DNA remains in solution. This allows separation of plasmids from the chromosome. Chromosomal DNA needs to be enzymatically digested into smaller fragments so they can enter the gel. Then the fragments can be resolved electrophoretically. DNA is negatively charged, and hence will migrate towards the positively charged pole along a gel matrix according to MW. Low-MW DNA fragments migrate faster and hence will appear as bands at lower positions in the lanes indicated in the figure. Sample 2 bacterial chromosomal DNA was digested with an enzyme that makes smaller fragments and a greater number of fragments than the enzyme used to cut sample 1 chromosomal DNA; hence, there are more bands in lane 2 of sample 2. See Fig. 2.3 for the use of restriction endonucleases. (Illustration by Ariana Reynolds.)

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2
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Image of Figure 2.3
Figure 2.3

Restriction endonuclease digestion of DNA. The restriction endonuclease BamHI recognizes a 6-base sequence ggatcc (1) and cuts between the two “g's,” generating two DNA fragments (2). If, in the same segment, the nucleotide “t” in ggatcc is replaced with “a,” then the enzyme BamHI will no longer recognize this target sequence (3) and hence the DNA fragment will remain intact (4). However, another enzyme, HindIII, recognizes the sequence aagctt and cuts the fragment between the two “a's” (5). Thus, HindIII generates two pieces of DNA of different MW.

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2
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Image of Figure 2.4
Figure 2.4

REA-Southern blot hybridization of DNA. Chromosomal DNA is extracted, purified (1), and digested with restriction endonuclease PvuII (2). The DNA fragments are then resolved by agarose gel electrophoresis (called restriction endonuclease analysis, or REA) (3). Each lane represents a different strain of . At this step, the REA resolution is poor because of the large number of DNA fragments generated by PvuII. The resolved DNA fragments are transferred onto a piece of nylon (or nitrocellulose) membrane (4). Here, the buffer in the tray flows upward by capillary action and elutes DNA fragments from the agarose gel, which then become fixed onto the membrane. The membrane containing the DNA fragments is probed with the IS probe (hybridization step) (5). The sharp black bands represent a copy of the IS in the chromosome. Thus, a gel image uninterpretable by REA becomes interpretable after the hybridization step. (Photographs courtesy of Lucilaine Ferrazoli of the Adolfo Lutz Institute, São Paulo, Brazil. Illustration by Ariana Reynolds.)

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2
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Image of Figure 2.5
Figure 2.5

Conventional gel electrophoresis and PFGE of DNA fragments in agarose gel. In PFGE, large pieces of DNA (>25 kb) “squeeze through” the gel matrix as the orientation of the electrical field across the gel is pulsed in different directions. The polarity of the electrical field is indicated by arrows. (Illustration by Ariana Reynolds.)

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2
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Tables

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Table 2.1

Bacterial pathogens for which complete genome sequences for more than one independent intraspecies strains have been reported or are about to be completed (as of 2002)

Citation: Riley L. 2004. Laboratory Methods Used for Strain Typing of Pathogens: Conventional and Molecular Techniques, p 29-62. In Molecular Epidemiology of Infectious Diseases. ASM Press, Washington, DC. doi: 10.1128/9781555817688.ch2

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