Selection of optimal conditions of PCR setup to identify canine brucellosis causative agent

Abstract

Canine brucellosis is a zoonotic disease that has a negative impact on the reproductive system of dogs and leads to significant economic losses of breeding kennels. The disease also poses a threat to human health, especially for veterinarians and breeders. Diagnostic tests for brucellosis are mostly based on serological and phenotypic methods which are quite laborious and have low specificity. The most advanced methods include molecular genetic approaches, in particular, PCR varieties based on bioinformatics assay. In this study, we have carried out assay of whole genome sequences of Brucella genomes presented in the GenBank database. Based on the results of the assay, loci were selected, some of which were present only in representatives of the species B. canis, others in B. abortus, and others in B. melitensis. Based on the selected DNA markers, primers and TagMan probes to allow detection and differentiation of brucellosis pathogen species were designed. Also, the optimal composition of the reaction mixture was selected and uniform amplification conditions were determined allowing PCR to be carried out under the conditions of one reaction. When performing multiplex PCR with DNA of strains of various species, it was found that oligonucleotide primers designed for amplification of representatives of the species B. abortus and B. melitensis had 100% specificity. In turn, primers designed to detect the main causative agent of canine brucellosis (B. canis) are also successful for amplification of B. suis bacteria. Thus, the developed primers and TagMan probes may be included in the real-time PCR diagnostic kit, suitable not only for indicating the main type of the causative agent of canine brucellosis - B. canis, but also “atypical” for these animals species B. suis, B. abortus and B. melitensis.