Capsule Typing of C. jejuni Isolates

Capsule typing was performed on genomic DNA extracts of C. jejuni isolates with four multiplex primer sets using 36 specific primers targeting capsule genes as developed and PCR reactions developed by Poly et al.^{26 (link),27 (link)}. Two µL of each C. jejuni isolate DNA was subjected to each multiplex PCR in a 25 µL reaction mixture containing 1X PCR buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl), 2.0 mM MgCl2, 300 µM concentration of each dNTPs (deoxynucleotide triphosphate), 0.4 µM of each primers sets (Alpha, Beta, Gamma and Delta) and 2.5 U of AmpliTaq Gold DNA polymerase. Amplification steps were as follows: 94 °C for 5 min; 28 cycles of 94 °C for 1 min, 52 °C for 1 min and 72 °C for 1 min and a final extension step at 72 °C for 10 min. Amplicons were visualized after gel electrophoresis at 120 V for 1 hour on a 2.0% Agarose-1000 gel (Invitrogen, USA) for Beta and Gamma set and 2.5% Agarose-1000 gel for Alpha and Delta set and staining with ethidium bromide. DNA of C. jejuni of known capsule types and 2-log DNA ladder (New England BioLabs, USA) were used as positive controls and a size marker, respectively.