|
Multilocus sequence typing (MLST) is a technique in molecular biology for the typing of multiple loci. The procedure characterizes isolates of microbial species using the DNA sequences of internal fragments of multiple housekeeping genes. Approximately 450-500 bp internal fragments of each gene are used, as these can be accurately sequenced on both strands using an automated DNA sequencer. For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST). The first MLST scheme to be developed was for ''Neisseria meningitidis'', the causative agent of meningococcal meningitis and septicaemia. Since its introduction for the research of evolutionary history, MLST has been used not only for human pathogens but also for plant pathogens. To assist the gathering and formatting of the utilized sequences a simple and free plug-in for Firefox has been developed ((link )). == Principle of MLST == MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles. The principle of MLST is simple: the technique involves PCR amplification followed by DNA sequencing. Nucleotide differences between strains can be checked at a variable number of genes depending on the degree of discrimination desired. The workflow of MLST involves: 1) data collection, 2) data analysis and 3) multilocus sequence analysis. In first section, definitive identification of variation is obtained by nucleotide sequence determination of gene fragments. In data analysis all unique sequences are assigned allele numbers and combined into an allelic profile and assigned a sequence type (ST). If new alleles and STs are found, they are stored in database after verification. In the final section of MLST the relatedness of isolates are made by comparing allelic profiles. Researchers do epidemiological and phylogenetical studies by comparing STs of different clonal complexes. A huge set of data is produced during the sequencing and identification process so bioinformatic techniques are used to arrange, manage, analyze and merge all of the biological data. To strike the balance between the acceptable identification power, time and cost for the strain typing, about seven to eight house-keeping genes are commonly used in the laboratories. Quoting ''Staphylococcus aureus'' as an example, seven housekeeping genes are used in MLST typing. These genes include carbamate kinase (''arcC''), shikimate dehydrogenase (''aroE''), glycerol kinase (''glpF''), guanylate kinase (''gmk''), phosphate acetyltransferase (''pta''), triosephosphate isomerase (''tpi'') and acetyl coenzyme A acetyltransferase (''yqiL'') as specified by the MLST website. However, it is not uncommon for up to ten housekeeping genes to be used. For ''Vibrio vulnificus'', the housekeeping genes used are glucose-6-phosphate isomerase (''glp''), DNA gyrase, subunit B (''gyrB''), malate-lactate dehydrogenase (''mdh''), methionyl-tRNA synthetase (''metG''), phosphoribosylaminoimidazole synthetase (''purM''), threonine dehyrogenase (''dtdS''), diaminopimelate decarboxylase (''lysA''), transhydrogenase alpha subunit (''pntA''), dihydroorotase (''pyrC'') and tryptophanase (''tnaA''). Thus both the number and type of housekeeping genes interrogated by MLST may differ from species to species. For each of these housekeeping genes, the different sequences are assigned as alleles and the alleles at the loci provide an allelic profile. A series of profiles can then be the identification marker for strain typing. Sequences that differ at even a single nucleotide are assigned as different alleles and no weighting is given to take into account the number of nucleotide differences between alleles, as we cannot distinguish whether differences at multiple nucleotide sites are a result of multiple point mutations or a single recombinational exchange. The large number of potential alleles at each of the loci provides the ability to distinguish billions of different allelic profiles, and a strain with the most common allele at each locus would only be expected to occur by chance approximately once in 10,000 isolates. Despite MLST providing high discriminatory power, the accumulation of nucleotide changes in housekeeping genes is a relatively slow process and the allelic profile of a bacterial isolate is sufficiently stable over time for the method to be ideal for global epidemiology. The relatedness of isolates is displayed as a dendrogram constructed using the matrix of pairwise differences between their allelic profiles, (eBURST ) or a minimum spanning tree (MST). The dendrogram is only a convenient way of displaying those isolates that have identical or very similar allelic profiles that can be assumed to be derived from a common ancestor; the relationships between isolates that differ at more than three out of seven loci are likely to be unreliable and should not be taken to infer their phylogeny. The MST connects all samples in such a way that the summed distance of all branches of the tree is minimal. Alternatively, the relatedness of isolates can also be analysed with MultiLocus Sequence Analysis (MLSA). This does not use the assigned alleles, but instead concatenates the sequences of the gene fragments of the housekeeping genes and uses this concatenated sequence to determine phylogenetic relationships. In contrast to MLST, this analysis does assign a higher similarity between sequences differing only a single nucleotide and a lower similarity between sequences with multiple nucleotide differences. As a result, this analysis is more suitable for organisms with a clonal evolution and less suitable for organisms in which recombinational events occur very often. It can also be used to determine phylogenetic relationships between closely related species. The terms MLST and MLSA are very often considered interchangeable. This is however not correct as each analysis method has its distinctive features and uses. Care should be taken to use the correct term. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「multilocus sequence typing」の詳細全文を読む スポンサード リンク
|