Primers and PCR conditions for MLST of S. pneumoniae
The pneumococcal MLST scheme uses internal fragments of the following seven house-keeping genes:
- aroE (shikimate dehydrogenase)
- gdh (glucose-6-phosphate dehydrogenase)
- gki (glucose kinase)
- recP (transketolase)
- spi (signal peptidase I)
- xpt (xanthine phosphoribosyltransferase)
- ddl (D-alanine-D-alanine ligase)
The primer pairs used for the PCR amplification of internal fragments of these genes are:
aroE-up, 5'-GCC TTT GAG GCG ACA GC aroE-dn, 5'-TGC AGT TCA (G/A)AA ACA T(A/T)T TCT AA gdh-up, 5'-ATG GAC AAA CCA GC(G/A/T/C) AG(C/T) TT gdh-dn, 5'-GCT TGA GGT CCC AT(G/A) CT(G/A/T/C) CC gki-up, 5'-GGC ATT GGA ATG GGA TCA CC gki-dn, 5'-TCT CCC GCA GCT GAC AC recP-up, 5'-GCC AAC TCA GGT CAT CCA GG recP-dn, 5'- TGC AAC CGT AGC ATT GTA AC spi-up, 5'-TTA TTC CTC CTG ATT CTG TC spi-dn, 5'-GTG ATT GGC CAG AAG CGG AA xpt-up, 5'-TTA TTA GAA GAG CGC ATC CT xpt-dn, 5'-AGA TCT GCC TCC TTA AAT AC. ddl-up, 5'-TGC (C/T)CA AGT TCC TTA TGT GG ddl-dn, 5'-CAC TGG GT(G/A) AAA CC(A/T) GGC AT
PCR amplification is carried out on chromosomal DNA using an extension time of 30 seconds, and an annealing temperature of 50 °C, with 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.
The DNA fragments are purified and sequencing reactions are carried out, in each direction, using the primers that were used for the initial PCR amplification.