Optimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi

Abstract

As a result of the work performed, the conditions for setting up multiplex PCR with electrophoretic detection for the diagnosis of horse strangles were determined, allowing the identification and differentiation of S. equi subsp. equi in one reaction. It was found that the developed PCR protocol for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection in a “multiplex” format has a high specificity and does not lead to amplification of PCR products with DNA of closely related microorganisms, saprophytic microflora, and bacterial pathogens. The sensitivity of the protocols for the detection and species differentiation of S. equi subsp. equi with electrophoretic detection was assessed. Diluted DNA samples of two S. equi subspecies were used as objects of research: S. equi subsp. equi and S. equi subsp. zooepidemicus. DNA samples were diluted by two-fold dilutions, starting from a concentration of 5 ng (which corresponds to 2 million 170 thousand copies in the genomic equivalent) to 1.19 * 10-6 ng (which corresponds to 0.71 copies in the genomic equivalent). DNA detection limit for S. equi subsp. equi was 66 copies in genomic equivalent or 152 fg, DNA of S. equi subsp. zooepidemicus – 132 copies in genomic equivalent or 305 fg.