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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.
| Gene | Direction | Primer Sequence |
|---|---|---|
| abcZ | abcZoF: | GTTTTCCCAGTCACGACGTTGTATCGCTGCTGCCACTTTTATCCA |
| abcZoR: | TTGTGAGCGGATAACAATTTCTCAAGGTCGCCGTTTAGAG | |
| bglA | bglAoF: | GTTTTCCCAGTCACGACGTTGTAGCCGACTTTTTATGGGGTGGAG |
| bglAoR: | TTGTGAGCGGATAACAATTTCCGATTAAATACGGTGCGGACATA | |
| cat | catoF: | GTTTTCCCAGTCACGACGTTGTAATTGGCGCATTTTGATAGAGA |
| catoR: | TTGTGAGCGGATAACAATTTCAGATTGACGATTCCTGCTTTTG | |
| dapE | dapEoF: | GTTTTCCCAGTCACGACGTTGTACGACTAATGGGCATGAAGAACAAG |
| dapEoR: | TTGTGAGCGGATAACAATTTCATCGAACTATGGGCATTTTTACC | |
| dat | datoF: | GTTTTCCCAGTCACGACGTTGTAGAAAGAGAAGATGCCACAGTTGA |
| datoR: | TTGTGAGCGGATAACAATTTCTGCGTCCATAATACACCATCTTT | |
| ldh | ldhoF: | GTTTTCCCAGTCACGACGTTGTAGTATGATTGACATAGATAAAGA |
| ldhoR: | TTGTGAGCGGATAACAATTTTATAAATGTCGTTCATACCAT | |
| lhkA | lhkAoF: | GTTTTCCCAGTCACGACGTTGTAAGAATGCCAACGACGAAACC |
| lhkAoR: | TTGTGAGCGGATAACAATTTCTGGGAAACATCAGCAATAAAC |
For strains of lineage 3, we use the following primers in case of failure with the above primers:
| Gene | Direction | Primer Sequence |
|---|---|---|
| lhkA | lhkA-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