Multilocus Sequence Typing of Vibrio parahaemolyticus

MLST analysis was conducted via the V. parahaemolyticus MLST website and database (http://pubmlst.org/vparahaemolyticus/) (Jolley et al., 2004 (link)). PCR conditions were denaturation at 96°C for 1 min; primer (Table S1; synthesized by Sangon Biotech, Shanghai, China) 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. PCR was performed with a Bio- Rad PTC-200 Thermal Cycler (Bio-Rad, California, USA) according to the manufacturer's directions. PCR products were sequenced on a BGI instrument (Shenzhen, China). The alignments of these sequences were determined using BioEdit. The sequences were analyzed online (http://pubmlst.org/vparahaemolyticus/) to assign allele numbers and define STs. New sequences for alleles and new ST profiles were submitted to the V. parahaemolyticus MLST database.
The evolution tree of the concatenated sequences of the seven loci was built based on the method of the Kimura-2-parameter in Mega 6.0 (Tamura et al., 2013 (link)). The ratio between the number of synonymous and nonsynonymous substitutions, showing the type of selection at each locus, was calculated using the method of Nei and Gojobori in Mega 6. The hypotheses of neutrality (dS = dN), purifying selection (dS/dN >1), and positive selection (dS/dN < 1) were tested using DNAsp 5.10 (Lüdeke et al., 2015 (link)).