φx174 hae 3 digest

The virulence genes analysed were hla, tst, pvl, fnbA and mecA, that codifies for α-haemolysin, toxic shock syndrome toxin, Panton Valentine leucocidin, fibronectin binding protein A and methicillin resistance, respectively. The analyses were carried out by partial PCR amplification of the genes in all strains, under the conditions described in S1 Table [27 (link)]. To detect amplified genes, PCR products were resolved using electrophoresis in 1.2% agarose gel (D1 Low EEO, Conda, 8018). The reference controls used to detect each virulence gene were: CECT 976 for hla, CECT 957 for tst, CECT 435 for lukS/F-PV, CECT 976 for fnbA and CECT 4439 for mecA, all from the Spanish Type Culture Collection (Spanish initials: CECT). In addition, a molecular weight standard (φX174-Hae III digest, Takara Bio Inc., 3405A) was included to determine the size of the amplified fragments.
On other hand, SCCmec typing was performed for positive mecA strains, using multiplex PCR described previously [28 (link)]. Furthermore, the amplified fragment was sequence and compared with Genbank database.
A relationship between MLST STs and the presence of virulence genes was investigated using Canoco software (version 5.12), conducting a constrained redundancy analysis (RDA).

PpIX was purchased from MP Biomedicals (Illkirch, France). Aminophenyl fluorescein (APF) was purchased from Goryo Chemical (Sapporo, Japan). Dihydroethidium (DHE), dimethyl sulfoxide (DMSO), methanol, ethanol, PI, and PBS buffer were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). SOD was purchased from MP Biomedicals (Eschwege, Germany). φX174 Hae III digest and pBR322 (Accession number: GenBank J01749.1) were purchased from Takara Bio (Kusatsu, Japan). A DNA damage detection kit (containing γH2AX monoclonal antibody and secondary antibody) was purchased from Dojindo Laboratories (Kumamoto, Japan).