Development of a Differential PCR Assay for Detection of Brucella abortus and Brucella melitensis: an Analytical Approach for Monitoring of Brucella spp. in Foods of Animal Origin

Background: Classical bacteriological detection of Brucella species from food, and environment is routinely carried out based on morphological and biochemical characteristics. However, for increasing specificity and sensitivity of species identification methods, development of a molecular assay is necessary that was main aim of this study. Methods: Panel of some reference strains belonging to the phylum Proteobacteria were specifically used in this study. Additionally, the panel was enriched with 20 Brucella field strains isolated from 13 cattle and 7 sheep (West Kazakhstan region), and six strains from three cattle and three sheep (Almaty region). Bacterial identification before designing was carried out based on 16S rRNA sequencing with universal primers. Primer design was implemented using the Primer3 program. Finally, specificity and sensitivity of the PCR assay for Brucella identification were evaluated. Results: The sensitivity of the developed conventional PCR assays was assessed with the range of 2×105 to 12 genomic copies isolated from B. abortus 100 and B. melitensis H-12 reference strains. The sensitivity of the developed assays using Ba and Ba-r, Bm and Bm-r primers was determined to be 1.6×103 genomic copies. Conclusion: Quick detection and species identification of Brucella strains circulating in Kazakhstan would help local authorities in decision-making and implementation of the most effective strategies for control of these bacteria. Our PCR-based assay was the first step towards developing a novel kit with final aim of standardizing molecular identification of B. abortus and B. melitensis in foods of animal origin in Kazakhstan and other central Asia countries.