The S. aureus MLST scheme uses internal fragments of the following seven house-keeping genes:
- arcC (Carbamate kinase)
- aroE (Shikimate dehydrogenase)
- glpF (Glycerol kinase)
- gmk (Guanylate kinase)
- pta (Phosphate acetyltransferase)
- tpi (Triosephosphate isomerase)
- yqi (Acetyle coenzyme A acetyltransferase)
Staphylococcus aureus DNA extraction protocol
- Resuspend 1/8 of a plate of overnight confluent growth from a blood agar plate in 400 µl lysis solution (see below).
- Incubate at 37 °C 30 min.
- Heat to 95 °C for 10 min.
- Place on ice and add 1 ml phenol/chloroform isoamyl alcohol (25:24:1) and mix thoroughly by inversion.
- Centrifuge 10,000 g for 20 min.
- Recover aqueous layer to fresh tube and precipitate DNA with the addition of 1 ml absolute ethanol.
- Place on ice for 15 min then pellet DNA by centrifugation at 10,000g for 20 min.
- Resuspend DNA in 50 µl water.
- 0.5 ml 5000 units/ml lysozyme
- 0.5 ml 500 units/ml lysostaphin
- 0.2 ml 0.5M EDTA
- 0.1 ml 1M Tris
- 8.7 ml de-ionised water
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.
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'