Primers and cycling conditions for Vibrio parahaemolyticus

Genes

The Vibrio parahaemolyticus MLST scheme uses internal fragments of the following seven house-keeping genes and the variable gene ompK:

Chromosome I:

recA (RecA protein)
dnaE (DNA polymerase III, alpha subunit)
gyrB (DNA gyrase, subunit B)

Chromosome II:

dtdS (Threonine dehyrogenase)
pntA (Transhydrogenase alpha subunit)
pyrC (Dihydroorotase)
tnaA (Tryptophanase)

PCR amplification

PCR amplification was carried out as follows (using the following primers that contain attached M13 forward and reverse primers, underlined). PCR conditions were denaturation at 96°C for 1 min, primer annealing at 58°C for 1 min, and extension at 72°C for 1 min, for 30 cycles, with a final extension step at 72°C for 10 min. Reagents concentration per reaction tube: 1.5 mM MgCl₂, 0.125 mM dNTPs (Qiagen), 0.5 mM each primer, 1 U Taq polymerase (Taq Platinum High Fidelity, Invitrogen). DNA used was 1ng per reaction tube.

Sequencing

The sequencing was conducted using M13 primers (forward and reverse).