Genes
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 |