IS711 sequencing of Brucella melitensis and Brucella abortus strains, and use of microchip-based real-time PCR for rapid monitoring

Abstract

In animal production systems around the world, brucellosis is a serious zoonotic disease that creates public health hazards and losses in economic terms. The aim of the study is to genotype and molecularly characterize Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) collected from different animal species and humans. A total of 50 isolates of Brucella species (16B. melitensis and 34B. abortus) were isolated from 1081 animal and human samples using a culture technique, followed by biochemical identification using the Vitek 2 compact system and proteomic identification using mass spectrometry technology. Molecular genotyping was performed on all isolates using multiplex real-time PCR. Six isolates from each genotype of Brucella species were selected and genetically evaluated by IS711 insertion sequences. Microchips-based real-time PCR for Brucella species identification was performed on twelve genetically characterized isolates as a first attempt. Forty-four (88%) isolates of Brucella species were detected using multiplex real-time PCR. Based on IS711 nucleotide sequencing, twelve isolates were phylogenetically clustered into their specific clusters. The results of the comparative analysis of conventional real time and microchips-based real time indicated that the later is faster and qualitatively more sensitive than conventional real time; however, further studies are needed to ensure that it is capable of serving as a gold standard alternative for Brucella species monitoring.

Keywords

• Brucella species
• Phylogenetic analysis
• microchip-based real-time PCR
• IS711 sequencing