Molecular Epidemiology of Microorganisms Methods and Protocols
The development of molecular genotyping methods has revolutionized the possibility for classification of microorganisms at the sub-species level. This investigation of species diversity is crucial for deciding the molecular relatedness of isolates for epi
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1. Introduction DNA fingerprinting or genotyping of Mycobacterium tuberculosis complex (MTB) strains became a priority in the United States when in the early 1990s a staggering increase in cases of multidrug-resistant tuberculosis (TB) was observed in New York City (1). Epidemiologists needed to know which cases were linked and where transmissions were occurring. They also needed to determine the size of the outbreak and to try to prevent further D.A. Caugant (ed.), Molecular Epidemiology of Microorganisms, Methods in Molecular Biology, Vol. 551 DOI: 10.1007/978-1-60327-999-4_10, © Humana Press, a part of Springer Science + Business Media, LLC 2009
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transmissions. The primary genotyping method available at the time, insertion element (IS) 6110-based restriction fragment length polymorphism (RFLP) analysis (2), provided excellent differentiation but required specialized software for analysis of the data as well as relatively long turnaround times for reporting of the results. Weeks or months could be required for the level of growth in culture necessary for performance of RFLP analysis. Data analysis required specialized matching software and expert interpretation for relating similar, but not identical, patterns. Genotyping methods that could employ amplification of nucleic acids were assessed in efforts to develop an alternative to RFLP analysis. The first widely adopted polymerase chain reaction (PCR)-based method for genotyping was spacer oligonucleotide typing or spoligotyping. Kamerbeek et al. (3) described a reversehybridization protocol to assay for the presence or absence of 43 specific DNA spacer sequences in the direct repeat (DR) region that had been identified in the strains M. tuberculosis H37Rv and Mycobacterium bovis BCG (Fig. 1). The majority of the 43
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Preparation of MTB sample DNA extraction or heat-killed cell suspension
Spoligotyping membrane with spacer DNA sequences bound
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Sample is used as template for PCR amplification of the Direct Repeat (DR) region.
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Unbound sample removed & remaining sample biotin-labeled PCR products detected
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Primer drA is biotinlabeled for detection Biotin-labeled sample PCR products hybridized against bound spacers sequences.
Results analyzed
Fig. 1. Basis of the spoligotyping methodology. (a) A spoligotyping membrane. Dashed lines indicate the location of the bound polymorphic oligonucleotides, one corresponding to each of the 43 unique spacer sequences utilized in the assay. (b) The hybridization of the amplified samples (black bars) against each of the bound oligonuleotides. (c) The excess and nonspecifically bound sample is removed through a series of washes, and the remaining bound PCR products from the sample are detected. (d) A representation of the final results.
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spacers were present in both H37Rv and BCG, but spacers 20, 21, and 33–36 were not present in H37Rv, and spacers 3, 9, 16, and 39–43 were missing in strains of BCG. The DR region consists of a repeated 36-bp sequ
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