Multilocus sequence typing (MLST) is a routine bacterial typing method based on nucleic acid sequencing, developed from the multilocus enzyme electrophoresis (MLEE). By directly sequencing house-keeping genes that express metabolism-related enzymes in MLST, it is possible to characterize evolutionary and population biology and thus to achieve bacteria strain typing.

MLST usually requires sequencing of 400-600 bp within 6-10 house-keeping genes, and the sequence of each locus is assigned an allele number based on the chronological order of its discovery. The allele numbers of each strain are arranged in the specified order to form its allelic profile, which is also called the ST (sequence type) of the strain. Each ST represents a unique set of sequence information, and by comparing the STs, the relevance of the strains can be researched.

The analysis method of MLST: Primers were designed and used for PCR amplification and sequencing of house-keeping genes to derive allele values at each locus for each strain. Allelic profiles or sequence types (STs) were then identified. Based on the allelic profile, a phylogenetic tree was constructed for cluster analysis using methods such as matrix pair-wise differences. In practice, for bacteria with established MLST protocols, the typing protocols can be obtained directly from the MLST database.

MLST can accurately document variation at the bacterial gene level, and nucleic acid sequences are easily accessible.Therefore, the method has been used not only to bacterial epidemiology, population biology, pathogenicity and evolution, but also to trace the origin and spread of antimicrobial-resistant bacterial strains.