Development of a Real-time PCR using Fluorescent Hybridization Probes
Objective: This research aims at the domestic development and design of experimental series of assay for plague causative agent determination through the method of real-time multi-locus polymerase chain reaction (PCR) with fluorescent hybridization probes, conducting control tests for refinement of optimal conditions to obtain the maximum sensitivity and specificity of the preparation.
Method: The research group designed fluorescent probes and synthesized the primers with unique nucleotide sequences for detection Yersinia pestis bacteria based on the chromosomal gene YPO2088, plasmid genes pst and caf1, which determine the properties of pesticide activity and capsular antigen synthesis and accordingly are associated with causative agent pathogenicity.
Results: This paper presents the results of the research conducted by the molecular diagnostics and genetics laboratory of research center related to the development of an experimental series of domestically designed polymerase chain reaction assays for plague agent determination through the method of real-time PCR with fluorescent hybridization probes. Based on the selected marker genes, primers YPO2088 F/R, caf1 F/R and pst F/R were developed. To determine their specificity, 49 Y. Pestis strains isolated from various objects, 26 strains of closely related bacteria and 22 non-Yersinia strains were used, as well as 288 suspensions from organs of rodents, fleas and mites obtained from the territories with plague natural foci located in Kazakhstan.
Conclusion: The research results have revealed that primers and probes were specific for Y. pestis, which enables to differentiate them from closely related and heterologous bacterial species, determine the mono- and non-plasmid variants of plague microbe phenotype with assay sensitivity of 100 fg for YPO-2088 F/R primers, and 10 fg for caf1 F/R and pst F/R primers