PF8
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Primers used for MLST of Clostridium difficile
 

- Genes

The Clostridium difficile MLST scheme uses internal fragments of the following seven housekeeping genes:

  • aroE (shikimate dehydrogenase)
  • dutA (dUTP pyrophosphatase)
  • gmk (gualynate kinase)
  • groEL (60 kDa chaperonin)
  • recA (recombinase)
  • sodA (superoxide dismutase)
  • tpi (triosephosphate isomerase)

- Primers for PCR amplification

aroE-F : CTAGTAGGTGAAAAACTCTCTCA
aroE-R : ACTGGTGTAGCATTTAATATTATATC

dutA-F : CCTAATTTTGCTCACAAAGGT
dutA-R : AAATCCAGTTGAGCCAAACC

gmk-F : AGGTGCAGGAAAAGGTACTAT
gmk-R : TTCATATGCACAGCTAAATCTC

groEL-F : ATACTGAATTAGATGCTGTTGAAG
groEL-R : TCTGAAGTAGTTTGCTCTACTTG

recA-F : CCAGATACAGGTGAACAGGC
recA-R : TTTAACATTTTCTCTTCCTTGTCC

sodA-F : TATGCATATGATGCACTTGAACC
sodA-R : AAATAGTAAGCATGCTCCCAAAC

tpi-F : GCAGGAAACTGGAAAATGCATAA
tpi-R : CAGATTGGCTCATATGCAACAAC

- Sequencing primers

The same primers as for PCR are used.

- PCR conditions

The PCR mixtures were heated for 3 min at 95°C, then a touch-down procedure followed, consisting of 30 s at 95°C, annealing for 30 s at temperatures decreasing from 60°C to 50°C during the first 11 cycles (with 1°C decremental steps in cycles 1 to 11), and ending with an extension step at 72°C for 30 s. A total of 40 cycles were performed

- Allele templates

aroE (408 bp)

ATCCACAAGAGAGTATTCGAGATATTGAATATTGAAAGTGCTTATAAAAACTTTGAAATTTCAAAAGAAGATATAT CTAAGCTAGACGGTGCTATAAAATTATTAGGAATACAAGGGGTAAATGTTACTGTACCATATAAGGAGCGTATTAT GAAATATCTTGATTTTATATCACCAGAGGCAAAACGTATAGGAGCAGTTAATACAATTTTACTCAGAGAAAATATG TTATATGGTTACAATACAGATTACTTTGGTCTTGATAGTATGTTTAAAATGGCTAATATTGATGTTCAAGGAAAAG TAGCAGTTATATTAGGAACTGGAGGTGCTTCTAAAGCTGCTCTTACATATTTTATTGATTCTGGTATAGAAAAGCT TTATGTTTCAACTAGAAAAAAGGATGAT

dutA (309 bp)

GTTGTAATACCTCCTGGAGAGACTAAACTTATCAAAACTGGGATATGTATAGAGCTTCCTACTATGACAGAAGCGC AGGTAAGACCTAGAAGTGGGCTTGCCTTAAAGCATTCTGTTACTGTATTAAATACACCAGGAACTATTGACGAAGG ATACAGAGGAGAGCTTAAAATTATATTAATAAATCATGGTAAAAATGATTTTAAGGTTGAAAAGCATATGAAAATT GCTCAAATGATAGTAAAACCTATCTATGATATTAACATAGAAGAAGTAAAAGAATTAAGTGACTCCGAAAGAGGAA AAGGT

gmk (279 bp)

AGAAAACCCAGAACTGGAGAAGTTGATGGAGTAAATTATTTCTTCATAAGTAAGGAAAAGTTTGAAGAGATGATAG AAAAAGGTGAGTTTTTGGAGTATGCTCAAATTTATGATAATTTTTATGGAACTCCAAAAGCGGCAATAATGGAATG TTTAGAAAAAGGACAAGATGTATTACTGGAGATTGAAATGCAAGGAGCAAAACAAATAAAAGAAGTTTGCCCAGAG GGAGTATTTATATTTGTACTACCACCATCACTTAAAGAATTAAAAAATAGA

groEL (363 bp)

GAAGCAGTTTTAAATGACCCATATATATTAATTACTGATAAAAAAATATCTAACATACAAGAATTATTACCAGTTC TTGAACAAATAGTTCAACAAGGTAAAAAGCTATTGATAATAGCTGAAGATGTAGAAGGTGAAGCATTATCTACATT AGTAGTAAATAAATTAAGAGGAACATTTGATGTAGTAGCAGTTAAAGCTCCAGGCTTTGGAGATAGAAGAAAAGAA ATGCTTCAAGATATAGCAATACTTACAGGTGCTCAAGTAATATCAGAAGAATTAGGTTATGATTTAAAAGAAGCTG ATTTATCTATGCTAGGTAGAGCTTCATCTGTTAAGGTAACTAAAGAAAGTACTACAATA

recA (378 bp)

GGAGCGATAGATATAATAGTAATAGACTCAGTAGCAGCATTAGTTCCAAAGGCTGAAATAGATGGAGATATGGGTG ATTCTCATGTAGGTTTACAAGCTAGACTTATGTCACAAGCACTTAGAAAGTTAACTGGTTCAATTAAAAAATCAAA TTGTGTTGCTATATTTATAAACCAGTTAAGAGAGAAAGTAGGAATAATGTTTGGAAACCCAGAAACTACTACTGGA GGACGTGCACTAAAATTCTATTCATCAGTTAGATTGGATGTTAGAAAAATAGATACAATAAAACAAGGTGATAAAG TTATAGGTAGTAGAACTAGAGTTAAAGTTGTTAAAAACAAAGTAGCACCACCATTTAAGCAAGCTGAATTTGAT

sodA (408 bp)

CATCATGATAAGCATTATCAAGCTTATGTTGATAAATTAAATGCTGCTCTTGAAAAATATCCTGAGCTTTATAATT ATTCTTTATGTGAATTATTGCAAAATTTAGATTCTTTACCTAAAGATATTGCTACAACTGTAAGAAATAATGCAGG TGGAGCTTATAATCATAAATTCTTTTTTGATATAATGACGCCAGAAAAAACCATACCTTCTGAATCTTTAAAAGAA GCTATTGATAGAGACTTTGGTTCTTTTGAAAAATTTAAGCAAGAGTTCCAAAAATCTGCTTTAGATGTCTTTGGTT CTGGTTGGGCTTGGCTTGTAGCTACTAAAGATGGGAAATTATCTATTATGACTACTCCAAATCAGGATAGCCCTGT AAGTAAAAACCTAACTCCTATAATAGGA

tpi (393 bp)

GAAATTAAAGATAAAGTAAATTCTGATAAAGTAGAAGCTGTTATATGTGCACCATTTACTTTATTAAAGGATTTAA AAGAAGCTACTAAGGGTACAAATATAAAGATAGGTGCTCAAAATATGCACTTTGAAGAAAAAGGAGCATTTACAGG AGAAGTTTCACCTCTTATGTTAAAAGAAATAGATATGGACTATGTTGTAATAGGACATTCAGAAAGAAGACAATAC TTCAATGAAACTGATGAAACTGTAAACAAGAAAGTTTTAAAAGCATTAGAAGTAGGAATAGACCCAATATTATGTG TTGGAGAAACTTTAGAACAAAGAGAAGCTGGAAAAACTAAAGATGTTTGTAAAGTTCAAGTTGAAAAAGCTTTAGA AAATGTTCTTAAA
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