Primer premier software version 6

The phs operon, containing the phsA, phsB, and phsC genes, is essential for the generation of H₂S [25 (link)]. Primers for these three genes were designed using the Primer Premier Software version 6.0 (PREMIER Biosoft, Palo Alto, CA, USA) based on S. Choleraesuis strain SC-B67 (NC_006905.1) and are shown in S1 Table. The PCR conditions were 95°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 45 s, 72°C for 1 min, followed by an extension period of 10 min at 72°C, then cooling to 4°C. Sequence results were imported into DNAStar version 7.1.0 (Lasergene) to assemble the complete operon. We aligned all of the sequences using MEGA software version 5.2 to identify mutations within DNA and protein sequences.

Alignment of the conserved genomic regions was performed and a pair of chimeric primers was designed for each target virus using the Primer Premier Software version 6.0 (Premier Biosoft, Palo Alto, CA). Each primer contained a gene-specific sequence with a universal tag at the 5’-end. A pair of primers targeting the universal tag was used with the forward primer labeled with cy5 fluorescent dye. Information of all primers was listed in Table 1. All primers were synthesized by Sangon Biotech (Shanghai, China).