Multilocus Sequence Typing Service

Multilocus Sequence Typing (MLST) is a molecular biological approach for typing based on multiple loci sequencing to fulfill the identification and classification of certain microbes. Since first proposed by Maiden in 1998 for the analysis of natural variation of Neisseria meningitides, MLST database has been developing and supplementing over the past two decades, this approach is widely used as a portable tool for the identifying clonal relationships among multiple pathogens, environmental bacteria or eukaryotes.

Figure 1. The principle of MLST (Ruppitsch 2016)

Generally, 6 to 10 housekeeping genes, which contain 400~600 bp internal fragments respectively, are sequenced. The sequence of each site is assigned an allele number based on the time when it was discovered, ST (sequence type) is acquired according to the combination of housekeeping gene allele numbers, each ST is correspondent with a specific strain type, and the correlation between strains can be found whereby comparing STs, which indicates that closely related strains have the same ST or only a few different gene loci of ST and vice versa - at least 3 different gene loci of ST among unrelated strains. MLST involves designing primers of housekeeping gene for PCR amplification and sequencing to abtain numeric of alleles, followed by allelic profile or sequence types analysis. Then methods such as matching matrix (matrix pair-wise differences) are used according to the results above to proceed clustering analysis.

Application & Advantages

Earlier schemes were explored including serological typing that differentiating bacterial isolates via interaction between antibodies and antigens, which has drawbacks like reliance on few antigenic loci and unpredictable reactivities of antibodies with different antigenic variants, and several molecular typing approaches such as pulsed-field gel electrophoresis (PFGE), ribotyping, and PCR-based fingerprinting that have been proposed to determine the relatedness of pathogens, while could not provide much evolutionary analyses. While MLST, with higher discriminatory power, one particular type of bacteria can be divided into more subtypes, and the phylogenetic relationship between different STs and the associated diseases can be determined, which has widely been used for epidemiological monitoring and evolutionary studies of a variety of pathogens. With the development of sequencing method and analysis software, MLST has gradually become a routine typing method for bacteria and is increasingly used as a tool for international comparisons of strains with the capability for characterization of bacteria and eukaryotes.

Figure 2. MLST genome taxonomic coverage provided by

What we can offer

Creative Biogene is the premier institution offering professional and comprehensive MLST services. We have years of experience to meet your specific project needs in using the MLST technology to add value to your research project.

The workflow of our MLST service involves:

1) Data collection: definitive identification of variation is obtained by nucleotide sequence determination of gene fragments;

2) Data analysis: all unique sequences are assigned allele numbers and combined into an allelic profile and assigned a sequence type. If new alleles and STs are found, they are stored in the database after verification;

3) Multilocus sequence analysis: the relatedness of isolates is made by comparing allelic profiles;

4) Epidemiological and phylogenetical studies by comparing STs of different clonal complexes.

While in a newly designed MLST scheme, the general elements include:

1) Select the strain that has been preliminarily screened;

2) Select gene loci with unique characteristics and design primers for gene amplification and sequencing;

3) Collect bacterial samples on the basis of existing typing information or epidemiological data.

We provide schemes to characterize pathogens from bacteria (e.g. Campylobacter spp., Clostridioides difficile, Enterococcus faecalis, Helicobacter pylori, Salmonella spp.) to eukaryotes (e.g. Aspergillus fumigatus, Candida albicans, Clonorchis sinensis, Saprolegnia parasitica). Our capabilities include but are not limited to:

Standard MLST:

• wgMLST (whole genome MLST)
• HiMLST (high-throughput MLST)
• cgMLST (core genome MLST)
• MVLST (multi-virulence-locus sequence typing)
• Bacteriophage MLST
• Plasmid MLST
• Ribosomal MLST
• MLSA (Multilocus Sequence Analysis)

Simply let us know your research requirement. We will provide support for each step in your workflow and propose the best strategy for you.

1. Ruppitsch W. Molecular typing of bacteria for epidemiological surveillance and outbreak investigation. Journal of Land Management, Food and Environment. 2016, 67: 199-224.

For research use only.

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