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)

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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

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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

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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

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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.

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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