DEVELOPMENT OF A METHOD FOR SPECIES IDENTIFICATION OF BACTERIA OF THE GENUS CLOSTRIDIUM BY MULTIPLEX REAL-TIME PCR

The aim of the study is to develop a method for species identification of 7 species of clostridia using multiplex real-time PCR. The paper presents the results of the development of a multiplex real-time polymerase chain reaction (PCR) for species identification of seven species of cattle clostridia. The results showed that clostridiosis is widespread in livestock farms of the Siberian Region, manifests itself in various clinical forms and depends on the type of pathogens and their associations. Using classical bacteriological methods, 510 samples of biomaterial from animals from seven regions of Siberia were examined. As a result, 50 isolates of clostridia of various species were isolated and identified using biochemical methods. To confirm species identity, a multiplex real-time PCR was developed that allows detection and typing of seven species of clostridia: C. sporogenes, C. perfringens, C. sordellii, C. histolyticum, C. septicum, C. chauvoei and C.novyi in mixed and pure bacterial cultures with a high degree of specificity. The sensitivity of the developed PCR in the study of artificially synthesized positive control samples was 7.2∙10–3.2∙102 GE, and in the study of pure cultures – 102–103 CFU/ml. The specific reaction was only with clostridia of its own species, but not with clostridia of other species or other types of bacteria. High specificity of the use of real-time PCR was demonstrated, allowing for the simultaneous detection of clostridia in mixed and pure bacterial cultures, which makes it a promising method in the laboratory diagnostics of various forms of cattle clostridiosis.