Multilocus sequence typing (MLST) is an unambiguous procedure for characterising isolates of bacterial species using the sequences of internal fragments of (usually) seven house-keeping 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 house-keeping gene, the different sequences present within a bacterial species are assigned as distinct alleles and, for each isolate, the alleles at each of the seven loci define the allelic profile or sequence type (ST).
Each isolate of a species is therefore unambiguously characterised by a series of seven integers which correspond to the alleles at the seven house-keeping loci.
In MLST the number of nucleotide differences between alleles is ignored and sequences are given different allele numbers whether they differ at a single nucleotide site or at many sites. The rationale is that a single genetic event resulting in a new allele can occur by a point mutation (altering only a single nucleotide site), or by a recombinational replacement (that will often change multiple sites) - weighting according to the number of nucleotide differences between alleles would erroneously consider the allele to be more different than by treating the nucleotide changes as a single genetic event.
Most bacterial species have sufficient variation within house-keeping genes to provide many alleles per locus, allowing billions of distinct allelic profiles to be distinguished using seven house-keeping loci. For example, an average of 30 alleles per locus allows about 20 billion genotypes to be resolved.
MLST is based on the well established principles of multilocus enzyme electrophoresis, but differs in that it assigns alleles at multiple house-keeping loci directly by DNA sequencing, rather than indirectly via the electrophoretic mobility of their gene products.
The great advantage of MLST is that sequence data are unambiguous and the allelic profiles of isolates can easily be compared to those in a large central database via the Internet (in contrast to most typing procedures which involve comparing DNA fragment sizes on gels). Allelic profiles can also be obtained from clinical material by PCR amplification of the seven house-keeping loci directly from CSF or blood. Thus isolates can be precisely characterised even when they cannot be cultured from clinical material.
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