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Primers and PCR conditions for MLST of S. pneumoniae

The pneumococcal MLST scheme uses internal fragments of the following seven house-keeping genes:

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.

Citing the database

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This publication made use of the Streptococcus pneumoniae MLST website (https://pubmlst.org/ spneumoniae/) sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 4356
Profiles (MLST): 13640
Last updated: 2017-12-15

Isolate database
Isolates: 37672
Last updated: 2017-12-15