Primers used for MLST of Klebsiella pneumoniae

Genes

The Klebsiella pneumoniae MLST scheme uses internal fragments of the following seven housekeeping genes:

rpoB (beta-subunit of RNA polymerase)
gapA (glyceraldehyde 3-phosphate dehydrogenase)
mdh (malate dehydrogenase)
pgi (phosphoglucose isomerase)
phoE (phosphorine E)
infB (translation initiation factor 2)
tonB (periplasmic energy transducer)

PCR amplification and Sequencing

Two protocols are available.
Protocol 1 uses the original primers and conditions of our publication (Diancourt et al., 2005) ;
Protocol 2 is a simplified protocol that uses primers with universal sequencing tails, which allows to PCR all genes at the same temperature and to sequence all genes with the same forward and reverse sequencing primers.

Protocol 1 (Diancourt et al., 2005) :

rpoB : F : Vic3 5'-GGC GAA ATG GCW GAG AAC CA-3'
rpoB : R : Vic2 5'- GAG TCT TCG AAG TTG TAA CC-3'

gapA : F : 173 5'-TGA AAT ATG ACT CCA CTC ACG G-3'
gapA : R : 181 5'-CTT CAG AAG CGG CTT TGA TGG CTT-3'

mdh : F : 130 5'- CCC AAC TCG CTT CAG GTT CAG-3'
mdh : R : 867 5'-CCG TTT TTC CCC AGC AGC AG-3'

pgi : F : 1R 5'-GAG AAA AAC CTG CCT GTA CTG CTG GC-3'
pgi : R : 1F 5'-CGC GCC ACG CTT TAT AGC GGT TAA T-3'

phoE : F : 604.1 5'-ACC TAC CGC AAC ACC GAC TTC TTC GG-3'
phoE : R : 604.2 5'-TGA TCA GAA CTG GTA GGT GAT-3'

infB : 1F 5'-CTC GCT GCT GGA CTA TAT TCG-3'
infB :1R 5'- CGC TTT CAG CTC AAG AAC TTC-3'

tonB: 1F 5'-CTT TAT ACC TCG GTA CAT CAG GTT-3'
tonB: 2R 5'-ATT CGC CGG CTG RGC RGA GAG-3'

PCR amplification is performed at an annealing temperature of 50°C for all genes except for gapA (60°C) and tonB (45°C).

We use the PCR primers also for sequencing, except for gene infB for which primer infB2F is used instead of the forward PCR primer, and for pgi, for which primers pgi2F and pgi2R are used.

infB2F : ACT AAG GTT GCC TCC GGC GAA GC
pgi2F : CTG CTG GCG CTG ATC GGC AT
pgi2R : TTA TAG CGG TTA ATC AGG CCG T

Protocol 2 (with universal sequencing primers):

rpoB F : Vic3oF 5'- GTT TTC CCA GTC ACG ACG TTG TA GGC GAA ATG GCW GAG AAC CA-3'
rpoB : R : Vic2oR 5'- TTG TGA GCG GAT AAC AAT TTC GAG TCT TCG AAG TTG TAA CC-3'

gapA : F : 173oF 5'- GTT TTC CCA GTC ACG ACG TTG TA TGA AAT ATG ACT CCA CTC ACG G-3'
gapA : R : 181oR 5'- TTG TGA GCG GAT AAC AAT TTC CTT CAG AAG CGG CTT TGA TGG CTT-3'

mdh : F : 130 5'- GTT TTC CCA GTC ACG ACG TTG TA CCC AAC TCG CTT CAG GTT CAG-3'
mdh : R : 867 5'- TTG TGA GCG GAT AAC AAT TTC CCG TTT TTC CCC AGC AGC AG-3'

pgi : F : 1R 5'- GTT TTC CCA GTC ACG ACG TTG TA GAG AAA AAC CTG CCT GTA CTG CTG GC-3'
pgi : R : 1F 5'- TTG TGA GCG GAT AAC AAT TTC CGC GCC ACG CTT TAT AGC GGT TAA T-3'

phoE : F : 604.1 5'- GTT TTC CCA GTC ACG ACG TTG TA ACC TAC CGC AAC ACC CAG TTC TTC GG-3'
phoE : R : 604.2 5'- TTG TGA GCG GAT AAC AAT TTC TGA TCA GAA CTG GTA GGT GAT-3'

infB : 1F 5'- GTT TTC CCA GTC ACG ACG TTG TA CTC GCT GCT GGA CTA TAT TCG-3'
infB :1R 5'- TTG TGA GCG GAT AAC AAT TTC CGC TTT CAG CTC AAG AAC TTC-3'

tonB: 1F 5'- GTT TTC CCA GTC ACG ACG TTG TA CTT TAT ACC TCG GTA CAT CAG GTT-3'
tonB: 2R 5'- TTG TGA GCG GAT AAC AAT TTC ATT CGC CGG CTG RGC RGA GAG-3'

PCR amplification is performed at an annealing temperature of 50°C for all genes.

Universal sequencing primers
Forward strand : primer oF : GTT TTC CCA GTC ACG ACG TTG TA
Reverse strand : primer oR : TTG TGA GCG GAT AAC AAT TTC

Allele templates