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Staphylococcus aureus MLST primers

The S. aureus MLST scheme uses internal fragments of the following seven house-keeping genes:

Staphylococcus aureus DNA extraction protocol

  1. Resuspend 1/8 of a plate of overnight confluent growth from a blood agar plate in 400 µl lysis solution (see below).
  2. Incubate at 37 °C 30 min.
  3. Heat to 95 °C for 10 min.
  4. Place on ice and add 1 ml phenol/chloroform isoamyl alcohol (25:24:1) and mix thoroughly by inversion.
  5. Centrifuge 10,000 g for 20 min.
  6. Recover aqueous layer to fresh tube and precipitate DNA with the addition of 1 ml absolute ethanol.
  7. Place on ice for 15 min then pellet DNA by centrifugation at 10,000g for 20 min.
  8. Resuspend DNA in 50 µl water.

Lysis solution

PCR conditions

PCR amplification is carried out on chromosomal DNA using an extension time of 30 s, and an annealing temperature of 55 °C, with Qiagen Taq polymerase. As the same primers are used for amplification and sequencing, it is important that only a single DNA fragment is amplified in the initial PCR. This may involve some optimisation of the annealing temperature.

PCR/Sequencing primers

arc up - 5' TTG ATT CAC CAG CGC GTA TTG TC -3'

arc dn - 5' AGG TAT CTG CTT CAA TCA GCG -3'

aro up - 5' ATC GGA AAT CCT ATT TCA CAT TC -3'

aro dn - 5' GGT GTT GTA TTA ATA ACG ATA TC -3'

glp up - 5' CTA GGA ACT GCA ATC TTA ATC C -3'

glp dn - 5' TGG TAA AAT CGC ATG TCC AAT TC -3'

gmk up - 5' ATC GTT TTA TCG GGA CCA TC -3'

gmk dn - 5' TCA TTA ACT ACA ACG TAA TCG TA -3'

pta up - 5' GTT AAA ATC GTA TTA CCT GAA GG -3'

pta dn - 5' GAC CCT TTT GTT GAA AAG CTT AA -3'

tpi up - 5' TCG TTC ATT CTG AAC GTC GTG AA -3'

tpi dn - 5' TTT GCA CCT TCT AAC AAT TGT AC -3'

yqi up- 5' CAG CAT ACA GGA CAC CTA TTG GC -3'

yqi dn- 5' CGT TGA GGA ATC GAT ACT GGA AC -3'

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the Staphylococcus aureus MLST website (https://pubmlst.org/ saureus/) sited at the University of Oxford (Jolley et al. Wellcome Open Res 2018, 3:124 [version 1; referees: 2 approved]). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 598,674
Profiles (MLST): 5,527
Last updated: 2019-08-22

Isolate database
Isolates: 34,372
Last updated: 2019-08-22