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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):
Primers for PCR amplification (P1)
| Gene | Direction | Primer Sequence |
|---|---|---|
| rpoB | F:Vic3 | GGCGAAATGGCWGAGAACCA |
| R:Vic2 | GAGTCTTCGAAGTTGTAACC | |
| gapA | F:gapA173 | TGAAATATGACTCCACTCACGG |
| R:gapA181 | CTTCAGAAGCGGCTTTGATGGCTT | |
| mdh | F:mdh130 | CCCAACTCGCTTCAGGTTCAG |
| R:mdh867 | CCGTTTTTCCCCAGCAGCAG | |
| pgi | F:pgi1F | GAGAAAAACCTGCCTGTACTGCTGGC |
| vR:pgi1R | CGCGCCACGCTTTATAGCGGTTAAT | |
| phoE | F:phoE604.1 | ACCTACCGCAACACCGACTTCTTCGG |
| R:phoE604.2 | TGATCAGAACTGGTAGGTGAT | |
| infB | F:infB1F | CTCGCTGCTGGACTATATTCG |
| R:infB1R | CGCTTTCAGCTCAAGAACTTC | |
| tonB | F:tonB1F | CTTTATACCTCGGTACATCAGGTT |
| R:tonB2R | ATTCGCCGGCTGRGCRGAGAG |
Note: For gene tonB, you must use MgCl2 at 50 mM, whereas for all other genes the primers work with MgCl2 at 25 mM.
PCR conditions (P1)
PCR amplification is performed at an annealing temperature of 50°C for all genes except for gapA (60°C) and tonB (45°C).
PCR cycle:
- 1 cycle:
- 94° for 2 min
- 35 cycles:
- 94° for 20 s
- 50°C (or 60°C, or 45°C) for 30 s
- 72° for 30 s
- 1 cycle:
- 72° for 5 min
Sequencing (P1)
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: ACTAAGGTTGCCTCCGGCGAAGC
pgi2F: CTGCTGGCGCTGATCGGCAT
pgi2R: TTATAGCGGTTAATCAGGCCGT
Protocol 2 (with universal sequencing primers):
Primers for PCR amplification (P2)
| Gene | Direction | Sequence |
|---|---|---|
| rpoB | F:Vic3oF | GTTTTCCCAGTCACGACGTTGTAGGCGAAATGGCWGAGAACCA |
| R:Vic2oR | TTGTGAGCGGATAACAATTTCGAGTCTTCGAAGTTGTAACC | |
| gapA | F:gapA173oF | GTTTTCCCAGTCACGACGTTGTATGAAATATGACTCCACTCACGG |
| R:gapA181oR | TTGTGAGCGGATAACAATTTCCTTCAGAAGCGGCTTTGATGGCTT | |
| mdh | F:mdh130oF | GTTTTCCCAGTCACGACGTTGTA CCCAACTCGCTTCAGGTTCAG |
| R:mdh867oR | TTGTGAGCGGATAACAATTTCCCGTTTTTCCCCAGCAGCAG | |
| pgi | F:pgi1FoF | GTTTTCCCAGTCACGACGTTGTAGAGAAAAACCTGCCTGTACTGCTGGC |
| R:pgi1RoR | TTGTGAGCGGATAACAATTTCCGCGCCACGCTTTATAGCGGTTAAT | |
| phoE | F:phoE604.1oF | GTTTTCCCAGTCACGACGTTGTAACCTACCGCAACACCGACTTCTTCGG |
| R:phoE604.2oR | TTGTGAGCGGATAACAATTTCTGATCAGAACTGGTAGGTGAT | |
| infB | F:infB1FoF | GTTTTCCCAGTCACGACGTTGTACTCGCTGCTGGACTATATTCG |
| R:infB1RoR | TTGTGAGCGGATAACAATTTC CGCTTTCAGCTCAAGAACTTC | |
| tonB | F:tonB1FoF | GTTTTCCCAGTCACGACGTTGTACTTTATACCTCGGTACATCAGGTT |
| R:tonB2RoR | TTGTGAGCGGATAACAATTTCATTCGCCGGCTGRGCRGAGAG |
These new primers work with MgCl2 at 25 mM for all genes.
PCR conditions (P2)
PCR amplification is performed at an annealing temperature of 50°C for all genes.
PCR cycle:
- 1 cycle:
- 94° for 2 min
- 35 cycles:
- 94° for 30 s
- 50°C for 1 min
- 72° for 30 s
- 1 cycle:
- 72° for 5 min
Sequencing (P2)
Universal sequencing primers:
F:primer oF: GTTTTCCCAGTCACGACGTTGTA
R:primer oR: TTGTGAGCGGATAACAATTTC
PCR product sizes
gapA: 662 nt; infB: 462 nt; mdh: 756 nt; pgi: 566 nt (or a bit more); phoE: 602 nt; rpoB: 1075 nt; tonB: 539 nt
The above sizes are obtained when using protocol 1. Please add 44 nt if you are using the universal sequencing adapters.
Allele templates
rpoB (501 bp):
ACCCACTACGGTCGCGTATGTCCGATCGAAACGCCTGAAGGTCCGAACATCGGTCTGATT
AACTCCCTGTCCGTGTACGCGCAGACCAACGAATATGGCTTCCTTGAGACGCCGTATCGT
AAAGTGACCAACGGTGTGGTTACTGACGAAATTCACTACCTGTCTGCTATCGAAGAAGGC
AACTACGTTATCGCTCAGGCGAACTCCAACCTGGATGAAAACGGCCACTTCGTAGAAGAT
CTGGTTACCTGCCGTAGCAAAGGCGAATCCAGCTTGTTCAGCCGCGACCAGGTTGACTAC
ATGGACGTATCCACCCAGCAGGTGGTATCCGTCGGTGCGTCCCTGATCCCGTTCCTGGAA
CACGATGACGCCAACCGTGCATTGATGGGTGCGAACATGCAACGTCAGGCGGTTCCGACT
CTGCGCGCTGATAAGCCGCTGGTTGGTACCGGTATGGAACGTGCTGTTGCCGTTGACTCC
GGTGTTACTGCCGTGGCTAAA
gapA (450 bp):
AACCTGAAGTGGGACGAAGTTGGTGTTGACGTTGTTGCTGAAGCAACCGGTATCTTCCTG
ACCGACGAAACCGCTCGTAAACACATCACCGCTGGCGCGAAAAAAGTCGTTCTGACTGGC
CCGTCCAAAGACAACACTCCGATGTTCGTTCGCGGCGCTAACTTCGACGCTTACGCAGGC
CAGGACATCGTTTCCAACGCTTCCTGCACCACCAACTGCCTGGCGCCGCTGGCTAAAGTT
ATCAACGACAACTTCGGTATCGTTGAAGGCCTGATGACCACCGTCCACGCTACCACCGCT
ACTCAGAAAACCGTTGATGGCCCGTCTCACAAAGACTGGCGCGGCGGCCGCGGCGCAGCT
CAGAACATCATCCCGTCCTCTACCGGCGCTGCTAAAGCAGTAGGTAAAGTACTGCCAGAA
CTGAACGGCAAACTGACCGGTATGGCGTTC
mdh (477 bp):
GGCGCGGATGTAGTGCTGATCTCCGCGGGCGTGGCGCGTAAGCCCGGCATGGATCGTTCC
GACCTGTTTAATGTGAATGCGGGTATCGTGAAGAACCTCGTGCAGCAGATTGCCAAAACC
TGCCCGCAGGCCTGCATCGGCATTATCACCAACCCGGTGAATACCACCGTGGCTATCGCC
GCCGAAGTACTGAAAAAAGCCGGCGTGTACGATAAAAACAAACTGTTCGGCGTTACCACG
CTGGACATCATCCGTTCCAATACCTTTGTGGCGGAGCTGAAAGGTAAATCGGCAACCGAG
GTGGAAGTCCCGGTCATTGGTGGTCACTCCGGGGTCACCATTCTGCCTTTACTGTCGCAG
ATCCCCGGCGTCAGCTTTAGCGATCAGGAAATTGCCGACCTGACTAAACGTATTCAGAAC
GCCGGTACCGAAGTCGTGGAAGCGAAAGCGGGCGGCGGGTCGGCGACCTTGTCGATG
pgi (432 bp):
GAGTCCAACGGTAAGTATGTTGACCGTAACGGCCACGCGGTAGACTACCAGACTGGCCCA
ATCATCTGGGGTGAGCCGGGCACCAACGGTCAGCACGCGTTCTACCAGCTGATCCACCAG
GGCACCAAAATGGTACCGTGCGATTTCATCGCTCCGGCTATCACCCACAACCCGCTGTCT
GACCACCATCAGAAACTGCTGTCTAACTTCTTCGCCCAGACCGAGGCCCTGGCCTTTGGT
AAATCCCGCGAAGTGGTTGAGCAGGAATATCGCGATCAGGGTAAAGACCCGGCGACCCTG
GAGCACGTGGTGCCGTTCAAAGTGTTCGAAGGTAACCGCCCGACTAACTCCATCCTGCTG
CGTGAGATCACCCCGTTCAGCCTCGGGGCGCTGATTGCCCTGTACGAGCACAAAATCTTC
ACCCAGGGCGCG
phoE (420 bp):
GTCGGCACCTCGTTAAGCTATGATTTCGGCGGCAGCGACTTCGCCGTCAGCGCAGCCTAC
ACCAGCTCCGACCGTACCAACGATCAGAACCTGCTGGCCCGCGGCCAGGGTTCGAAAGCG
GAAGCCTGGGCGACCGGCCTGAAATATGACGCCAACAATATCTACCTGGCGACCATGTAC
TCTGAAACCCGCAAGATGACCCCGATCAGCGGCGGCTTTGCCAACAAAGCGCAGAACTTT
GAAGCGGTGGCGCAGTATCAGTTCGACTTCGGTCTGCGTCCGTCCCTCGGCTATGTGCTG
TCGAAAGGGAAGGATATCGAAGGGGTGGGGAGTGAAGATCTGGTTAACTACATCGACGTG
GGCCTGACCTACTACTTCAACAAAAACATGAACGCCTTCGTGGATTACAAAATCAACCAG
infB (318 bp):
GGCATGATCACCTTCCTGGATACCCCGGGCCACGCCGCGTTTACCTCCATGCGTGCTCGT
GGCGCGCAGGCGACGGATATCGTGGTTCTGGTGGTGGCGGCAGACGACGGCGTGATGCCG
CAGACTATCGAAGCTATCCAGCACGCTAAAGCGGCGCAGGTACCGGTGGTAGTGGCGGTG
AACAAGATCGATAAGCCAGAAGCCGATCCGGATCGCGTGAAGAACGAACTGTCCCAGTAC
GGCATCCTGCCGGAAGAGTGGGGCGGCGAGAGCCAGTTCGTCCACGTTTCCGCGAAAGCG
GGTACCGGCATCGACGAC
tonB (414 bp, except some variants with a few codons deleted to inserted):
ATGGTGGCGCCGGCCGATCTTGAGCCGCCTCCGGCGGCGCAGCCTGTCGTGGAGCCCGTT
GTTGAACCCGAACCTGAGCCGGAGCCAGAGGTAGTGCCTGAACCGCCGAAAGAGGCGCCG
GTGGTGATCCATAAACCGGAACCTAAGCCGAAGCCCAAACCTAAACCCAAGCCTAAGCCG
GAGAAAAAGGTTGAACAGCCGAAGCGGGAAGTGAAGCCGGCAGCAGAGCCGCGTCCGGCC
TCGCCGTTTGAAAACAACAATACGGCGCCGGCGCGTACAGCGCCAAGCACCTCGACAGCA
GCGGCTAAACCCACCGTTACTGCTCCAAGCGGCCCGCGGGCGATCAGCCGCGTTCAGCCG
TCCTATCCAGCGCGCGCTCAGGCGCTGCGCATTGAAGGTACGGTACGGGTGAAG