Protocols used for MLST of Yersinia pseudotuberculosis¶
Genes¶
The Y. pseudotuberculosis MLST scheme uses internal fragments of the following seven house-keeping genes:
- adk (adenylate kinase)
- argA (N-acetylglutamate synthase)
- aroA (3-phosphoshikimate-1-carboxylvinyltransferase)
- glnA (glutamine synthase)
- thrA (aspartokinase I/homoserine dehydrogenase I)
- tmk (thymidylate kinase)
- trpE (anthranilate synthase component I)
PCR Amplification¶
The primer pairs we use for the PCR amplification of internal fragments of these genes are:
| Product length: | ||
| adk_P1: | F 5’-ATGCGTATCATTCTGCTGGG-3’ | 641 bp |
| adk_P2: | R 5’-CCGAGAATAGTCGCCAGTTC-3’ | |
| argA_P1: | F 5’-GGATTTCGCCACTCAGTTCC-3’ | 615 bp |
| argA_P2: | R 5’-ATCAGTCACCCCTTGTGATG-3’ | |
| aroA_P3: | F 5’-AGCGGCCAATTGGCCATTTG-3’ | 802 bp |
| aroA_P4: | R 5’-CACATCGCCATACGGTGGTC-3’ | |
| glnA_P1: | F 5’-GCTGACTTCTTCGAAGAAGG-3’ | 701 bp |
| glnA_P2: | R 5’-GACATATGGCAGTGCATACC-3’ | |
| thrA_P3: | F 5’-CGTCTTTGCGGTGATGTCG-3’ | 823 bp |
| thrA_P2: | R 5’-GTTGGTGTCATACAAGAATTTACG-3’ | |
| tmk_P3: | F 5’-TATTGAAGGGCTTGAAGGGG-3’ | 606 bp |
| tmk_P4: | R 5’-TGATTGGTCAGCCACTGAGC-3’ | |
| trpE_P3: | F 5’-CACCAGTTACAACAAGCGCC-3’ | 743 bp |
| trpE_P4: | R 5’-GTATCCAAATCACCGTGAGC-3’ |
An annealing temperature of 55° C is fine for all genes.
Sequencing¶
adk_S1: F 5’-TGGAGAAATACGGCATTCCG-3’ adk_S2: R 5’-ACATTACGGGTTCCGTCCAG-3’
argA_S3: F 5’-CAAGACATTTGTTGTCATGC-3’ argA_S4: R 5’-ATGGCTAACTGAGTTGCAAC-3’
aroA_S3: F 5’-TGCACAGATCGATTATCTGG-3’ aroA_S2: R 5’-ATGGTCATTGCAGCATCAGG-3’
glnA_S1: F 5’-TTTGATGGCTCCTCGATTGGTG-3’ glnA_S2: R 5’-TTGGTCATGGTATTGAAGCG-3’
thrA_S1: F 5’-GATGTGATGGAACATCTGGC-3’ thrA_S2: R 5’-GTCACCACATGGAAGCCATC-3’
tmk_S5: F 5’-GGCCCAAGGGATTAACGATAT-3’ tmk_S2: R 5’-AATGGGTTGGGAGGCATCAAT-3’
trpE_S1: F 5’-CCAGAGATGGCGTTACAGTG-3’ trpE_S4: R 5’-TAGCCCACTGCACCGCCGTA-3’
Allele template¶
Allelic profile of Y. pseudotuberculosis strain SP93422.
adk (389 bp):
AAAGCAGGTTCTGAGTTAGGTCTGAAAGCAAAAGAAATTATGGATGCGGGCAAGTTGGTGACTGATGAGTTAGTTATCGCAT
TAGTCAAAGAACGTATCACACAGGAAGATTGCCGCGATGGTTTTCTGTTAGACGGGTTCCCGCGTACCATTCCTCAGGCAGAT
GCCATGAAAGAAGCCGGTATCAAAGTTGATTATGTGCTGGAGTTTGATGTTCCAGACGAGCTGATTGTTGAGCGCATTGTCGG
CCGTCGGGTACATGCTGCTTCAGGCCGTGTTTATCACGTTAAATTCAACCCACCTAAAGTTGAAGATAAAGATGATGTTACCG
GTGAAGAGCTGACTATTCGTAAAGATGATCAGGAAGCGACTGTCCGTAAGCGTCTTAT
argA (361 bp):
CTAGTATCGTCAACGATATAGGCTTATTACATAGCCTTGGGATCAGATTAGTCGTGGTATACGGTGCCCGACCACAGATTGAC
AGTAATCTGGCAGATCATAACTACGAACCTATTTATCATAAACATACCCGAGTGACTGACGCCCGGACTCTGGAGATGGTCA
AGCAAGCGGCCGGTTTATTACAACTGGATATCACGGCCAGGCTTTCAATGAGTTTGAACAACACCCCACTGCAAGGTGCTCAT
ATCAACGTAGTTAGCGGTAACTTTATCATTGCTCAACCGCTAGGTGTGGATGATGGTGTTGATTATTGCCATAGCGGGCGTATC
CGGCGCATTGATGAAGAAGCTATTCATCG
aroA (357 bp):
TTGATGGGCGTGTCTCTAGCCAGTTCCTGACTGCTTTATTGATGACCGCCCCGCTGGCGGAGCAAGATACGACTATTCGGATT
TGGGTGATCTGGTTTCCAAACCTTATATCGATATTACTCTGCACTTGATGAAAGCATTTGGTATTGACGTGGGGCATGAGAAC
TACCAAATTTTCCACATCAAAGGGGGTCAGACCTACCGCTCACCAGGGACTTATTTGGTTGAGGGCGATGCCTCGTCGGCTTC
CTACTTCTTAGCGGCTGCGGCTATTAAGGGGGGAACAGTGCGTGTCACTGGTATTGGCAAGAAAAGTGTACAGGGCGACACT
AAATTTGCCGATGTGTTGGAAAAAA
glnA (338 bp):
CACTGATTATCCGTTGTGACATTCTTGAGCCAGGCACCATGCAGGGCTATGACCGCGATCCACGTTCTATCTCCAAACGTG
TGAAGACTTCCTGAAATCTTCAGGTATCGCTGATACCGTGTTGTTCGGGCCAGAACCAGAATTCTTCCTGTTTGACGATATC
CGTTTTGGTAGCAGCATCAATGGGTCACACGTATCCATCGATGACGTCGAAGGCGCATGGAACTCCAGCACCAAATACGA
AGGCGGCAACAAAGGCCACCGTCCTGCAGTAAAAGGCGGTTACTTCCCTGTTCCTCCGGTCGACTCTGCACAAGACCTCC
GTTCTGCCATGTGT
thrA (342 bp):
TCAATATTATTGCTATCGCTCAAGGCTCGTCTGAGCGCTCTATTTCTGTGGTTGTCAATAATGATGCGGTCACTACCGGGGTGC
AGTTTGCCACCAGATGCTGTTTAACACGGATCAAGTCATTGAGGTGTTTGTCATTGGCGTGGGTGGTGTGGGGGGCGCATTAA
TCGAACAAATCTATCGCCAGCAACCGTGGTTGAAACAGCGTCATATCGATTTACGTGTTTGTGGTATCGCGAATTCCAAAGCC
ATGTTGACCAACGTGCATGGCATTGCATTAGATAACTGGCGTCAGGAACTGGCTGAAGTTCAGGAGCCGTTTAACCTGAGCC
GCTTGATCC
tmk (375 bp):
AAAACTGCGTGATTTGATTAAGCAAGGTATTGACGGTGAGGTCCTGACGGATAAAGCCGAGGTATTAATGCTGTATGCCG
CCAGGGTGCAATTAGTAGAAAATGTCATCAAGCCAGCACTGGCGCGTGGCAGTTGGGTTGTGGGTGACCGCCATGATTTG
TCATCACAGGCATATCAGGGCGGGGGGCGAGGGATTGACAGCCAACTGATGGCCTCGTTACGTGACACGGTGTTAGGCG
AATTCCGGCCAGACCTGACCTTATATTTGGATCTGCCACCGGCTGTGGGCTTGGCGCGCGCGCGCGCGCGCGGTGAATTGG
ACCGTATCGAACAAGAGTCACTGGCGTTTTTTGAGCGAACCCGCGCACGTTACCT
trpE (465 bp):
CCATCCCGCCGTTTTACGCTGCCCTGCCCATCACCGCTGGCGGCCTATCAGACACTGAAAGACCATAATCCCAGCCCCTAC
ATGTTTTTCATGCAAGACAATGATTTTTCTCTGTTTGGCGCATCACCTGAAAGCGCACTGAAATACGATGCCAGCAACCGT
CAAATTGAGATTTACCCGATTGCGGGTACTCGTCCACGCGGTCGTCGTCCTAATGGAGAACTCGATCGTGATTTAGACAG
CCGTATCGAGTTGGAAATGCGTACTGACCATAAAGAGATGGCAGAACATTTAATGTTGGTGGATCTGGCTCGTAACGATC
TGGCACGTATTTGCGAACCCGGTAGCCGCTATGTTGCAGATTTAACCAAAGTTGACCGTTACTCTTTTGTCATGCATCTGG
TGTCCCGCGTGATTGGCACTCTGCGTCAAGATTTAGATGTGCTGCATGCTTATCAAGCGTGT