DNA Sensor for the Detection of Brucella spp. Based on Magnetic Nanoparticle Markers.

Due to the limitations of conventional Brucella detection methods, including safety concerns, long incubation times, and limited specificity, the development of a rapid, selective, and accurate technique for the early detection of Brucella in livestock animals is crucial to prevent the spread of the associated disease. In the present study, we introduce a magnetic nanoparticle marker-based biosensor using frequency mixing magnetic detection for point-of-care testing and quantification of Brucella DNA. Superparamagnetic nanoparticles were used as magnetically measured markers to selectively detect the target DNA hybridized with its complementary capture probes immobilized on a porous polyethylene filter. Experimental conditions like density and length of the probes, hybridization time and temperature, and magnetic binding specificity, sensitivity, and detection limit were investigated and optimized. Our sensor demonstrated a relatively fast detection time of approximately 10 min, with a detection limit of 55 copies (0.09 fM) when tested using DNA amplified from Brucella genetic material. In addition, the detection specificity was examined using gDNA from Brucella and other zoonotic bacteria that may coexist in the same niche, confirming the method's selectivity for Brucella DNA. Our proposed biosensor has the potential to be used for the early detection of Brucella bacteria in the field and can contribute to disease control measures.

Genomic distribution of the insertion sequence IS711 reveal a potential role in Brucella genome plasticity and host preference.

The Insertion Sequence 711 (IS711) is linked to the Brucella genus. Mapping the genomic distribution of IS711 can help understand this insertion element's biological and evolutionary role. This work aimed to delineate the genomic distribution of the IS711 element and to study its association with Brucella evolution. A total of 124 genomes representing 9 Brucella species were searched using BLASTn sequence alignment tool to identify complete and truncated copies of IS711. Based on the genomic context, each IS711 locus was assigned a code using the initial letters of its neighboring genes. Various tools were used to annotate the neighboring genes and determine the shared synteny around orthologous IS711 loci. The tool Islandviewer 4 was used to scan for genomic islands. The Codon Tree method was used to build phylogenetic trees of B. melitensis, B. abortus, and B. suis genomes. The phylogenetic trees of the three species were analyzed, taking into account the genomic distribution patterns of IS711. The result of IS711 frequency analysis showed a relatively conserved number of copies/genome for the different species and for some biovars. The analysis showed that Brucella species with a relatively low IS711 copy number (4-8 copies/genome) are linked to domestic animals as primary hosts and have potential for zoonotic transmission. However, species with a relatively higher copy number (12-30 copies/genome) are less zoonotic and tend to be linked with wild animals as primary hosts. Analyzing the genomic distribution map of IS711 loci showed several unique patterns of IS711 distribution that are correlated with the evolution of Brucella species and biovars. The results also showed that 46.2% of the conserved IS711 elements are located within genomic islands. Based on our results and previous data, we postulate a model explaining the IS711 role in Brucella evolution. We assume that during the transition from a free-living to an intracellular lifestyle, a descendant of the Brucella genus had acquired a progenitor sequence of the IS711. Subsequently, a burst in IS711 transposition occurred. This parasitic expansion can be deleterious and has to be counteracted by evolutionary forces to prevent lineage extension and to promote adaptation to host. Similar to other plasmid-free pathogenic α-Proteobacteria bacteria, the balance of expansion and reduction of insertion elements could be one of the mechanisms to control genome reduction and streamlining. We hypothesize that the IS711-mediated genomic changes and other small sequence nucleotide changes in specific orthologous genes could significantly contribute to Brucella's evolution and adaptation to different animal hosts.