Multiplex PCR Assay for Serotyping

Almost all primers and probes (Table 2) for MSA were designed based on the wzx or wzy gene using Primer Premier v5.0 software (Premier Biosoft International, Palo Alto, CA, United States), except those targeting orf10_type8, which is utilized to differentiate type 8 from type 5. Multiplex PCR amplification was performed in a 50 μL reaction mixture composed of 100 ng genomic DNA, 1 × Goldstar PCR buffer, 0.04 mM deoxynucleoside triphosphates, 0.1 mM each primer, and 1 unit Goldstar DNA polymerase. The PCR parameters were as follows: 95°C for 10 min; 30 cycles at 98°C for 10 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. Probe-microsphere coupling, hybridization and MSA analysis were performed as described previously (Guo et al., 2018 (link)). A positive signal was defined as a median fluorescence intensity (MFI) ≥ 200 and a signal/background ratio (S/B ratio = MFI/Blank) > 5.