Open AccessOptimization of Conditions for The Multiplex PCR for Diagnostics of Horse Strangles with Subspecies Differentiation of Streptococcus Equi Subsp Equi
Author(s) -
Kalysh Berdimuratova,
R. Makhamed,
Alexandr Shevtsov
Publication year - 2020
Publication title -
eurasian journal of applied biotechnology
Language(s) - English
Resource type - Journals
eISSN - 2617-1147
pISSN - 2617-1139
DOI - 10.11134/btp.2.2020.8
Subject(s) - streptococcus equi , biology , multiplex polymerase chain reaction , genomic dna , multiplex , polymerase chain reaction , dna , microbiology and biotechnology , horse , bacteria , genetics , gene , paleontology
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.