Molecular Serotyping of Actinobacillus pleuropneumoniae

Molecular serotyping was carried out according to the method described by Bossé et al. [11 (link)]. For better differentiation of the PCR products in the agarose gel, this procedure was applied in three sequential steps. Step I included testing for serovars 1, 2, 10, 13 & 14; step II for serovars 3, 5, 7, 12, 15, 16 & nadV and step III for serovars 4, 6, 8, 9/11, 17 & 18. The respective reference strains of A. pleuropneumoniae were used as positive controls as well as the reference strain for serovar 13 as control for the detection of nadV. PCR were carried out using Qiagen HotStart Taq DNA Polymerase (Qiagen, Hilden, Germany) and the standard reaction set up for a 25 µl reaction volume including a final concentration of 1.5 mM MgCl₂ and 200 µM of each dNTP (Carl Roth, Karlsruhe, Germany). Primers (Biomers, Ulm, Germany) were used at a final concentration of 0.3 µM each. Cycling conditions were 15 min at 95 °C for activation of Taq, 30 s at 95 °C for denaturation, 90 s at 56 °C for annealing and 2 min at 72 °C for elongation. A final extension step at 72 °C for 10 min was programmed and a total of 30 cycles were run on a Bio-Rad T100 cycler (Bio-Rad, Munich, Germany). 16 µl PCR product was run in a 2% agarose gel for 90 min for pattern analysis.