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For citation please refer to: Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L.H., Karch, H., Reeves, P. R., Maiden, M. C., Ochman, H., and Achtman M. 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol.Microbiol. 60(5), 1136-1151.
Update information: ST complexes have been updated again on 17.05.2007. There are currently 600 STs and 54 ST complexes. The criteria have also been changed and are now groups of at least 3 STs sharing 6 alleles in pair-wise comparisons. The assignments of STs to some of the previous ST complexes have changed as a result, although we have tried to maintain consistency.
ST complexes have been updated again on 24.08.2005. Multiple new ST Complexes have been assigned and multiple STs have been assigned to known complexes. Due to the increased number of strains assigned to the ST29 Complex, it has become unclear whether these bacteria are closely related or only linked by one intermediate recombinant. Therefore, this has now been split into the ST23 and ST29 Complexes.
ST complexes have been updated on 23.11.2004. This includes the merging of ST21, 29 and 90 Complexes into ST29 Complex and ST3 and 17 Complexes into ST20 Complex. Multiple new ST complexes have been assigned. A number of STs have been merged with other STs due to curation of the database.
Protocols used for MLST of Escherichia coli
Genes
The E. coli MLST scheme uses internal fragments of the following seven house-keeping genes:
PCR Amplification
Please note: These include new primer sequences (added 26 July 2004; labelling corrected on 5 March, 2007) whose labels indicate the genomic direction rather than reading frame. The primer pairs for the PCR amplification of internal fragments of these genes can be chosen from:
| Gene | Primer pair sequences | Product length | Annealing Temperature |
| adk | F 5'-ATTCTGCTTGGCGCTCCGGG-3' R 5'-CCGTCAACTTTCGCGTATTT-3' F1 5'-TCATCATCTGCACTTTCCGC-3' R1 5'-CCAGATCAGCGCGAACTTCA-3' |
583 bp | 54° C |
| fumC | R1 5'-TCCCGGCAGATAAGCTGTGG-3' F 5'-TCACAGGTCGCCAGCGCTTC-3' R 5'-GTACGCAGCGAAAAAGATTC-3' |
806 bp | 54° C |
| gyrB | F 5'-TCGGCGACACGGATGACGGC-3' R1 5'-GTCCATGTAGGCGTTCAGGG-3' R 5'-ATCAGGCCTTCACGCGCATC-3' |
911 bp | 60° C |
| icd | F 5'-ATGGAAAGTAAAGTAGTTGTTCCGGCACA-3' R 5'-GGACGCAGCAGGATCTGTT-3' |
878 bp | 54° C |
| mdh | F 5'-ATGAAAGTCGCAGTCCTCGGCGCTGCTGGCGG-3' R 5'-TTAACGAACTCCTGCCCCAGAGCGATATCTTTCTT-3' F1 5'-AGCGCGTTCTGTTCAAATGC-3' R1 5'-CAGGTTCAGAACTCTCTCTGT-3' |
932 bp | 60° C |
| purA | F1 5'-TCGGTAACGGTGTTGTGCTG-3' F 5'-CGCGCTGATGAAAGAGATGA-3' R 5'-CATACGGTAAGCCACGCAGA-3' |
816 bp | 54° C |
| recA | R1 5'-AGCGTGAAGGTAAAACCTGTG-3' F 5'-CGCATTCGCTTTACCCTGACC-3' F1 5'-ACCTTTGTAGCTGTACCACG-3' R 5'-TCGTCGAAATCTACGGACCGGA-3' |
780 bp | 58° C |
Conditions:
PCR: 2 min at 95°, 30 cycles of 1 min at 95°, 1 min at annealing temp, 2 min at 72° followed by 5 min at 72°. The PCR reaction contains 50 ng of chromosomal DNA, 20 pmol of each primer, 200 umol (10 ul of a 2 mM solution) of the dNPTs, 10 ul of 10x PCR buffer, 5 units of Taq polymerase and water to 100 ul.
Sequencing
We use the amplification primer pairs for the sequencing step.
Allele template
Allelic profile of E. coli strain MG1655 (see Genebank)
Updated