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Primers used for MLST of Listeria monocytogenes

Genes

The Listeria monocytogenes MLST scheme uses internal fragments of the following seven housekeeping genes:

abcZ (ABC transporter)
bglA (beta glucosidase)
cat (catalase)
dapE (succinyl diaminopimelate desuccinylase)
dat (D-amino acid aminotransferase)
ldh (L-lactate dehydrogenase)
lhkA (histidine kinase)


PCR amplification

Protocol uses primers with universal sequencing tails to sequence all genes with the same forward and reverse sequencing primers.

GeneDirectionPrimer Sequence
abcZabcZoF:GTTTTCCCAGTCACGACGTTGTATCGCTGCTGCCACTTTTATCCA
abcZoR:TTGTGAGCGGATAACAATTTCTCAAGGTCGCCGTTTAGAG
bglAbglAoF:GTTTTCCCAGTCACGACGTTGTAGCCGACTTTTTATGGGGTGGAG
bglAoR:TTGTGAGCGGATAACAATTTCCGATTAAATACGGTGCGGACATA
catcatoF:GTTTTCCCAGTCACGACGTTGTAATTGGCGCATTTTGATAGAGA
catoR:TTGTGAGCGGATAACAATTTCAGATTGACGATTCCTGCTTTTG
dapEdapEoF:GTTTTCCCAGTCACGACGTTGTACGACTAATGGGCATGAAGAACAAG
dapEoR:TTGTGAGCGGATAACAATTTCATCGAACTATGGGCATTTTTACC
datdatoF:GTTTTCCCAGTCACGACGTTGTAGAAAGAGAAGATGCCACAGTTGA
datoR:TTGTGAGCGGATAACAATTTCTGCGTCCATAATACACCATCTTT
ldhldhoF:GTTTTCCCAGTCACGACGTTGTAGTATGATTGACATAGATAAAGA
ldhoR:TTGTGAGCGGATAACAATTTTATAAATGTCGTTCATACCAT
lhkAlhkAoF:GTTTTCCCAGTCACGACGTTGTAAGAATGCCAACGACGAAACC
lhkAoR:TTGTGAGCGGATAACAATTTCTGGGAAACATCAGCAATAAAC

For strains of lineage 3, we use the following primers in case of failure with the above primers:

GeneDirectionPrimer Sequence
lhkAlhkA-F3:GCAAGTTTTGAATACGTATCAGCG
lhkA-R2:TACGCATTTCATGAGAAACATCAG

- Universal sequencing primers

oF : GTT TTC CCA GTC ACG ACG TTG TA

oR : TTG TGA GCG GAT AAC AAT TTC

PCR conditions

  • 1 cycle:
    • 94° for 4 min
  • 35 cycles:
    • 94° for 30 s
    • 52° for 30 s* (35 cycles)
    • 72° for 2 min
  • 1 cycle:
    • 72° for 10 min

*PCR amplification is performed at an annealing temperature of 52°C for all genes except for bglA (45°C).


Allele templates

abcZ (537 bp):
AAATCGACGAACAGAATGCGTATAGGACTTTTCCGCAAGATGGAAAAACTATCAATCCGT
TTCTTCGATAGTCGCAATGATGGCGAAATGCTTAGTCGTTTCACTAGTGACTTAGATAAT
ATTTCCAACACACTAAACCAAGCATTGATCCAAGTACTATCCAACGTCGCGCTAATGATT
GGTGTTATCATCATGATGTTCCAACAAAACGTGGAACTAGCCTTCGTTACTCTAATATCT
GCTCCATTTGCGATTATTATTGCGACAGTGATTATTCGAAAAGCCCGCAAATTCGTTGAT
ATTCAACAAGATGAACTAGGCGTACTTAACGGCTACATTGACGAAAAAATCTCTGGTCAA
AAAATTATTATCACAAATGGCTTAGAAGAAGAAACAATTGACGGCTTTGTTAAACAAAAC
AATATCGTTAAAAACGCAACTTACAAAGGTCAAGTTTACTCCGGTTTACTTTTCCCAATG
ATGCAAGGTATTTCCCTATTAAATACAGCTATTGTTATCTTCTTCGGTGGATGGTTA

bglA (399 bp):
AACCAATTCGAAGGCGCTTACAACGTCGATGGAAAAGGACTTTCCGTTCAAGATGTTACT
CCAAAAGGCGGATTTGGTCACATTACTGACGGTCCAACACCAGATAACTTAAAATTAGAA
GGAATTGACTTCTATCATCGCTACAAAGATGACGTGAAACTTTTTGCCGAAATGGGCTTC
AAGGTTTTCCGTACTTCCATCGCTTGGTCCCGTATCTTCCCAAATGGTGACGAAACTGAG
CCAAACGAAGCAGGATTACAATTTTACGATGATTTATTCGATGAACTTCTAGCACATAAT
ATCGAACCACTGATTACTTTATCTCACTATGAAACACCACTTCACTTATCGAAAACTTAC
GACGGCTGGGTAAATAGAAAAATGATCGACTTCTATGAA

cat (486 bp):
GCTCGTGGTGCTGGTGCTCACGGGAAATTTGTCACTAAGAAAAGCATGAAAAAATATACA
ATGGCTAAATTTTTGCAAGAAGAAGGAACGGAAACGGAGGTTTTTGCTCGTTTTTCAACA
GTAATTCATGGGCAACATTCTCCAGAAACATTACGTGATCCACGCGGTTTCTCCGTTAAG
TTTTATACAGAAGAAGGGAATTATGATTTTGTCGGAAATAATTTGCCGGTATTCTTCATT
CGTGATGCGATTAAGTTTCCGGATGTTATTCATTCCTTGAAGCCTGATCCACGCACAAAT
ATTCAAGATGGCAACCGTTACTGGGATTTCTTTAGCCTTACACCGGAAGCTACGACGATG
ATTATGTACTTATTCAGTGATGAAGGAACGCCGGCTTCTTACCGGGAAATACGTGGCTCT
AGTGTTCATGCGTTCAAATGGATTAATGAAGAAGGCAAAACAGTTTATGTAAAACTGCGC
TGGATT

dapE (462 bp):
TTGCAAAAGTTGTTAGCTGAACACGGTATTGAGTCCGAAAAGGTAAAATACGACGTAGAC
AGAGCCAGCCTAGTTAGCGAAATTGGTTCCAGTGACGAGAAAGTTTTGGCGTTTTCAGGG
CATATGGATGTCGTTGATGCGGGTGATGTCTCGAAGTGGAAGTTCCCACCTTTTGAAGCA
ACAGAGCATGAAGGGAAAATATACGGACGTGGCGCGACGGATATGAAGTCAGGTCTAGCG
GCGATGATTATTGCAATGATTGAGCTTCATGAAGAAAAACAAAAACTAAATGGCAAAATT
AGATTATTAGCAACGGTTGGTGAAGAAGTCGGTGAACTTGGAGCCGAACAACTAACGCAA
AAAGGTTACGCAGATGATTTAGATGGCTTGATTATCGGCGAACCGAGTGGACACCGGATT
GTTTATGCGCATAAAGGTTCCATTAATTATACCGTTAAATCC

dat (471 bp):
GAAGTAGTTCGTCTATATAATGGAAAATTCTTCACTTATAATGAACACATTGATCGTTTA
TATGCGAGTGCAGCAAAAATTGACTTAGTTATTCCTTATTCTAAAGAAGAGTTACGAGCG
TTACTTGAAAAATTAGTTGCTGAAAATAATATTAATACAGGAAATGTCTATTTACAAGTG
ACTCGAGGTGTTCAAAACCCGCGTAATCACGTTATGCCAGATGATTTCCCGCTGGAAGGC
GTTTTAACAGCAGCAGCTCGTGAAGTACCAAGAAATGAACAACAATTTGTGCAAGGTGGA
CCAGTAATTACAGAAGAAGATGTTCGTTGGTTACGCTGTGACATCAAGAGTTTGAATTTA
CTTGGAAACATTTTAGCAAAAAACAAAGCACATCAACAAAATGCGTTAGAAGCTGTTTTA
CACCGCGGAGAGCAAGTAACTGAGTGTTCAGCTTCCAATATTTCTATTATT

ldh (453 bp):
TATAGCGACTGCCACGATGCGGACTTAGTTGTTGTAACTGCCGGGACTGCACAAAAACCT
GGTGAAACTCGTTTAGATTTAGTAAATCGTAATATTAAAATCATGAAAGGCATCGTGGAT
GAAGTAATGGCTAGCGGATTTGACGGTATCTTCTTAATCGCTTCTAACCCAGTAGATATC
TTAACTTACGCTACATGGAAATTCTCAGGTCTTCCAAAAGAACGTGTTATCGGTTCTGGA
ACAAGCCTTGATACAGCACGTTTCCGTATGTCAATTGCCGACTATCTAAAAGTAGATGCT
CGTAACGTCCATGGTTACATCCTTGGCGAACACGGCGATACAGAATTCCCAGCATGGAGC
CACACAACTGTCGGCGGTCTTCCAATCACTGAATGGATTAGCGAAGATGAACAAGGTGCA
ATGGATACTATTTTCGTAAGTGTTCGTGATGCA

lhkA (480 bp):
TATCCAACACAGATGAATCAGCCGTTACCAAAGGATTTCTCTATTTCTTCGGATGATAAG
AAAAAACTTGAAAGTGGCGAAACAGTTAGTAAGAAAATAGATAATCGCTTTAATAAAGAA
ATGACAATTGTGTACGTCCCAATAATGAATGGCGACAAATTTGTCGGTTCTATCGTGCTC
AATTCACCTATTAGCGGTACGGAGCAAGTAATTGGTACGATTAACCGCTATATGTTCTAC
ACTATTTTACTTTCTATAACGGTAGCACTTATTCTTAGCGCAATCTTGTCCAAACTACAA
GTAAATCGAATCAACAAACTACGAGCAGCGACAAAAGACGTTATTCAAGGCAATTACAAA
GCTCGATTGAAGGAAAATAATTTTGATGAAATTGGTGCACTTGCCATTGATTTCAATAAA
ATGACACAAACCCTTGAAACATCTCAAGAAGAAATAGAACGACAAGAGAAGCGGAGACGC

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