Protocols used for MLST of Moraxella catarrhalis¶
Genes¶
The M. catarrhalis MLST scheme uses internal fragments of the following eight house-keeping genes:
- glyRS (glycyl-tRNA synthetase beta subunit)
- ppa (Pyrophosphate phospho-hydrolase)
- efp (elongation factor P)
- fumC (fumarate hydratase)
- trpE (anthranilate synthase component I)
- mutY (adenine glycosylase)
- adk (Adenylate kinase)
- abcZ (ATP-binding protein)
PCR Amplification¶
The primer pairs we use for the PCR amplification of internal fragments of these genes are:
| Product length: | ||
| glyRS: | F 5’-GCACCGAAGAGTTGCCACCA-3’ | 762 bp |
| glyRS: | R 5’-ACGCAACGGGCAAATCCACC-3’ | |
| ppa: | F 5’-AATAAAATTCTAGATGCTGGC-3’ | 523 bp |
| ppa: | R 5’-ACTTATTGCTCTGTCCAGCG-3’ | |
| efp: | F 5’-CTCTGATTGACAACTGGCAGG-3’ | 582 bp |
| efp: | R 5’-GATATTCGCCAGTACGCG-3’ | |
| fumC: | F 5’-GGGCGGTACAGCAGTCGGCAC-3’ | 675 bp |
| fumC: | R 5’-CTCATCAAATTCAGCTTCAG-3’ | |
| trpE: | F 5’-TTATCCCGCATCGAAAATGG-3’ | 545 bp |
| trpE: | R 5’-GGTTTCATCCCATTCAGCC-3’ | |
| mutY: | F 5’-GGCAATACCATCATCAGCCG-3’ | 609 bp |
| mutY: | R 5’-GGTAACTGACTTTGAACGCC-3’ | |
| adk: | F 5’-GGCATTCCTCAAATCTCAAC-3’ | 631 bp |
| adk: | R 5’-GATGGGCTTTATTGTCAAATG-3’ | |
| abcZ: | F 5’-ACATGCTGATGATGGTGAG-3’ | 610 bp |
| abcZ: | R 5’-CACTGGCAAGTTCAAGCGC-3’ |
An annealing temperature of 52° C is fine for all genes, except glyRS (58° C) and adk (54° C).
Sequencing¶
We use the amplification primer pairs for the sequencing step except for the following genes.
glyRS: F 5’-GCACCGAAGAGTTGCCACCA-3’ glyRS: R 5’-ATATCGGCTTGACGCTGATC-3’
fumC: F 5’-GCTGTCAAAGTCGCTAAAG-3’ fumC: R 5’-CTCATCAAATTCAGCTTCAG-
mutY: F 5’-TATGCTGTGTGGGTATCTG-3’ mutY: R 5’-GGTAACTGACTTTGAACGCC-3’
Allele template¶
Allelic profile of M. catarrhalis MC216
glyRS (537 bp):
GTTGGCACTTTACTAAATGATCAAAATATCAGCTTTGATGATATTCATGCCTTTGCCGCACCACGCCGTTTGGCACTTACTATTAC
AGGTGTGGGCGAGTTTCAGCCTGACCGTATCGAAACTAAAAAAGGCCCAAGTGTTAAGGCAGCGTTTGATGACGCAGGTAATTTAA
CGCGTGCAGGGCAAGGATTTTTGCAAGGTTTAAATGCCACTGGGCGAAATCTTAGTGTCAAAGATTTGGGGCGTATTGCTGATAAA
AAAGGCGAGTACATCGCTTATGATTTGACCATTAAAGGCGAAAAAATCACCGATTTAATGCCAAATATCTTACAAAAAGCGCTTGA
TGATTTGCCAATTGCTAAGCGTATGCGTTCTGGCATTGATCGCCATGAGTTCATACGGCCTGTTCATTGGGTGGTTTTGATGTCAG
ATGATGTTGTAATTGAGGCGACCATTCAAGGCCATAAAACTGGCAACCAAAGCCGTGGTCATCGCTATCATTCACCTGATTTTTTT
GCCATTGATCATGCCGATCAT
ppa (393 bp):
TCAAATCATAAAATTGAGTGGAATCGTCATTTGGCATGCTTTGAGCTTGATCGTGTTGAGCCAATTGCCTTTGCCAAACCATGCAA
CTATGGCTTTATTCCCCAAACCTTAGATGAAGATGGTGATGAATTAGACGCCTTAATCATCACAGAACAGCCTTTGACGACGGGTA
TTTTTCTTAAAGCCAAAGTCATCGGGGTAATGAAATTTGTGGATGATGGCGAGGTCGACGATAAAGTCATCGTTGTTCCTGCCGAT
GATCGTAATAATGGCAACGCCTATAATAGTCTTGACGACCTACCACCACAGTTAATAAAGCAACTTGAATTTCATTTTAGTCATTA
TAAAGACTTAAAAAAACCAGGCTCAACTGTGGTTGAGGGCTTTTTTGAT
efp (414 bp):
AATGAATATGTGAAACCAGGTAAAGGTCAAGCTTTTAACCGTGTTAAGTTGCGTAATCTACGCACAGGCAAAGTGCTTGAGCAGAC
TTTTAAGTCAGGCGATTCACTTGAAGGTGCTGATGTGGTGGATGTGGAAATGAACTATCTATACAACGATGGCGAATTTTGGCATT
TTATGCACCCTGAGACTTTTGAGCAATTGCAAGCAGATAAAACCGCAATGGGTGATGCTGCACAGTGGCTAAAAGAAAATTCAAAT
GCGTTATGCACTTTGACGCTATTTAATGGTGCGCCGCTGTCGGTCACACCACCAAACTTTGTAGAGCTTGAAATCGTTGAAACTGA
CCCAGGTGTTCGCGGTGATACCTCAGGTGGTGGCGGTAAGCCTGCACGCCTTGAAACAGGTGCAACCGTG
fumC (465 bp):
GAGGCTTTGGCCGCACGAGATGCCGAAGTATTTGCTTCAGGTGCACTAAAAACACTGGCAGCAAGCCTAAATAAAATCGCCAATGA
TGTGCGTTGGCTGGCTTCAGGCCCACGCTGCGGTCTAGGTGAGCTTAGCATTCCTGAAAATGAGCCTGGTTCATCCATCATGCCTG
GTAAAGTCAATCCAACTCAGTGTGAGGCGATGACCATGGTGGTCACCCAAGTCTTTGGTAATGATACGACCATTTCTATGGCAGGG
GCTTCTGGTAATTTTGAATTAAATGTTTATATGCCTGTGATTGGATATAACTTATTGCAGTCGATTCGTCTGCTAACGGATGCGAT
CAATAGCTTTAATGAGCATTGTGCGGTAGGGATCGAACCAATTTACGAAAAAATTGACCATTATTTACATCATTCTTTGATGTTGG
TCACCGCACTCAATCGCCATATTGGCTATGAAAAT
trpE (372 bp):
TTAAATGACCAAAAAGAGATCGCAGAACATCTGATGCTCATTGATCTTGGGCGTAATGATATTGGGCGTGTCTGTGAGATTGGTTC
GGTTAAAGTGACAGATCAGATGTTCATTGAACGCTACTCGCAAGTGATGCATATCGCTAGTAATGTTGAGGGGCGTATACGCCAAG
ATGTCGATGCTTTAGATGTGTTTTGTGCGACCTTTCCAGCGGGTACTTTGTCTGGTGCGCCAAAAATCCGTGCCATGCAAATTATT
GATGAAGTTGAGCCAGTGCGTCGCACCGTTTTTGGCGGGGCGATCGGATATCTTGGCTGGCATGGCAATATGGATACTGCCATCGC
AATACGCACAGCGGTCATGAAAGATGGT
mutY (426 bp):
TTTTTTGAGCCGTTTTTGGCACGATTTGCAACCGTACAAGAGCTTGCTGTAGCAGATTGGCAAGAAGTAGCGTCTTTTTGGGCAGG
GCTTGGGTATTATGCTCGGGCAAGAAATTTACACGCAGGCGCACAGCAAGTGGCTGATTTTATTGATACTCATGGCAGGTTTCCTG
AAACGGTCAATGAGTGGCAAGCAGTCAAAGGGGTGGGGCGATCGACAGCTGGCGCAATCGTTGCGATGGGCGTTAAGAAGTTTGGC
GTCATCTGCGATGGCAATGTCAAGCGTGTACTTGCTCGCCATCGCGCGGTTTTTGGTGATGTGACAAAAAGCGCAACTGACAAAAG
GCTTTGGGAAATTGCCACCGCACTAACCCCGAAAGAGTATAGTGGACACTATGCCCAAGCGATGATGGATTTGGGTGCAACA
adk (471 bp):
CAAGCAAAAAGTGTCATGGATGCAGGTCAATTGGTTTCTGATGAGCTGATTATTAATTTGGTTAAAGAACGCTTAAAACAGCCAGA
TTGTGACAATGGTTGTATTTTTGATGGATTCCCACGCACAATCCCACAAGCCAAAGCACTGGCAGATGCTGGCATTGCCATTGACC
ATGTGATTGAAATCAGCGCCCCTGATGATGAAATCGTAAGCCGTATGTCGGGTCGCCGTGCACATTTGGCATCAGGTCGTACTTAT
CATGTGATCTATAATCCCCCAAAAACTGACGGTATTGATGATGTCACAGGCGAGGCACTGGTGCAGCGTGATGATGATAAAGAAGA
AGTGGTGCGTGATCGCTTATCGGTATATCACGCCCAAACCGCTACATTGATTAGTCATTATCAAGAAGTTGCCAAATCGGGCGAAA
ATGCCCCTGAATATCATGACTTTGATGGAACAAAATCCATC
abcZ (429 bp):
CTTGATGTGGTGATGGAGGGTGATGACAAAGTTGCCCAAGCCTTAAAAAACTATCATCAAGCCAGTGCAAAATGCGCCATGGGCGA
CAATGAAGCTTGTGATGCCATGAGCGGTCTTCAGCAAACACTCGATGAGTTAAATGGTTGGGACTTGGAGCGGCGTGCGCGTGCAT
TGATTGACAGCATGGGGTTTGACGCTGATGCTTGCTTATCAACATTATCAGGGGGTCGTAAACGCCGTGTTCTGTTAGCACAGGCT
TTGGTAACAAATCCTGATGTGCTACTTTTAGACGAGCCAACCAACCATTTAGATATTGAAAGCATCACTTGGCTTGAAAAATTTTT
ATTAAATGAACGATTAACCGTTCTTTTCATCTCACATGATCGGAAGTTTGTTGATACACTTGCCACTTATATCATCGAGCTTGAC