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Primers and cycling conditions for Vibrio vulnificus MLST

The Vibrio vulnificus MLST scheme uses internal fragments of the following ten house-keeping genes:

glp (Glucose-6-phosphate isomerase)
gyrB (DNA gyrase, subunit B)
mdh (Malate-lactate dehydrogenase)
metG (Methionyl-tRNA synthetase)
purM (Phosphoribosylaminoimidazole synthetase)
dtdS (Threonine dehyrogenase)
lysA (Diaminopimelate decarboxylase)
pntA (Transhydrogenase alpha subunit)
pyrC (Dihydroorotase)
tnaA (Tryptophanase)

PCR amplification

PCR amplification was carried out as follows (using the following primers):

Reaction conditions were denaturation at 94°C for 1 min, primer annealing at 50°C for 45 sec, and extension at 72°C for 1 min, for 30 cycles.

Locus Name and sequence of primer Amplicon size (bp)
Forward (5' to 3') Reverse (5' to 3')
glp glp-A1, AAC GTT GAT GGT ACT CAC ATC G glp-A2, CAC AGC CGG TGC AAT GAA G 678
gyrB  gyrB-A1, GGT ACC ACG GTA CGT TTC TG gyrB-A2, CTT TAC GGC GTG TCA TTT CAC 700
mdh  mdh-A1, ATC CCA ACC CAC GTT TCG A mdh-A2, CTC ACC GTA AGG TAG GAT CTC T  702
metG  metG-A1, ACC TGC CTC AGT TTG AAA GC metG-A2, TGC TGC GGC AAC AAA TTC G 695
purM  purM-A1, GTG CTT TAT GCG AAC TAC CAA C purM-A2, CGG CCA TTC CCA GCT CTT TC 694
dtdS  dtdS-A1, GGT CAC AAA AAA CCA TCC CAG dtdS-A2, CAT TTC ACG GCC ATA GAT ACC 724
lysA  lysA-A1, GCA CTA CGC ATT AAA ACC GCT lysA-A2, GTT TTC TGC AAT CAC ATC CAC G 862
pntA  pntA-A1, GGC ACT GGA TGC GTT GTC pntA-A2, TGA AGA TTG AGT CGG CAA ACG 598
pyrC  pyrC-A1, CTC ATC ATG CCA AAC ACT GTC pyrC-A2, GCG TGA GCC GTG TAA GAG 698
tnaA  tnaA-A1, GGC GAA AAA CGT CTA CAC CG tnaA-A2, GGT GAA CTC GAG TCC TTT GAC 919

Sequencing

Sequencing was carried out as follows (with the following primers):

Reaction conditions were denaturation at 94°C for 10 sec, primer annealing at 50°C for 5 sec, and extension at 60°C for 2 min, for 30 cycles.

Locus Name and sequence of primer Amplicon size (bp)
Forward (5' to 3') Reverse (5' to 3')
glp glp-S1, AGT CGA TCC TGA GAC AAC ACT C glp-S2, GCC TTG GTG GAT CAG TTG GTA 480
gyrB  gyrB-S1, ACA TCC TAG CGA AGC GTC TAC G gyrB-S2, AGA ACA AAC GGT TTT CGC TTC G 459
mdh  mdh-S1, ACT AGA AGG TGC TGA TGT GGT T  mdh-S2, TGT GCA AAG AAG CTA GCA TGC T 489
metG  metG-S1, CGC TCT GGC TCT CTG CAA ACT G metG-S2, TTG CCT TCA AAG CGT TTC GCA A 429
purM  purM-S1, ACT GAT GGC GTT GGC ACC AAG purM-S2, CAG AAA CCG CCG CCA GTG 444
dtdS  dtdS-S1, ATT GGT GTG GGC GTA AAC CG dtdS-S2, CAT TTC ACG GCC ATA GAT ACC 417
lysA  lysA-S1, GGA GTT GGT CGC TTC TGA AG lysA-S2, GAT AAA GCG ACC CGG TTC AG 465
pntA  pntA-S1, GTT ATC GAG CGG TTG TTG AAG pntA-S2, CCG ATC ACT TTT ACG CCG TT 396
pyrC  pyrC-S1, GAT CGC ATT CAA GCA CAT AAC C pyrC-S2, GTC CGT ACC CAA AAA GAA CTT C 423
tnaA  tnaA-S1, ATC ATG GAC TCA(T) GCC CGT TT tnaA-S2, CTA AAG CAT GGG CAG GGA ACT 324

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the Vibrio vulnificus Multi Locus Sequence Typing website (https://pubmlst.org/ vvulnificus/) developed by Keith Jolley and sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:595). The development of this site has been funded by the Wellcome Trust.

Status

Sequence database
Sequences: 1007
Profiles (MLST): 349
Last updated: 2017-02-07

Isolate database
Isolates: 457
Last updated: 2017-02-07