Multiplex PCR Serotyping of Isolates

The isolates were further serotyped by the molecular method published by Howell et al., 2015, following modifications by Schuwerk et al., 2020 [12 (link)], which divided the assay into two sets of multiplex PCR (mPCR). The PCR reactions were set up consisting of 12.5 µL 2× Mytaq™ HS Red Mix PCR master mix (Meridian Bioscience^®, Cincinnati, OH, USA), 0.5 nM of forward and reverse primer each, 2 µL (50–100 ng/µL) template and nuclease-free water topped-up to a volume of 25 µL. The PCR assay was performed at 95 °C for 1 min, followed by 30 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s, and a final extension of 72 °C for 5 min. All PCR products were subjected to 2% agarose gel electrophoresis in TAE buffer 80 V for 45 min, using a 100-bp molecular weight marker (Qiagen, Hilden, North Rhine-Westphalia, Germany) as a guide. Electrophoresed gels were visualized using a UV transilluminator and gel documentation system (Syngene, Frederick, MD, USA). The PCR bands were measured using the Image Lab software v6.1 (Bio-rad Laboratories, Inc., Hercules, CA, USA).