Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei

Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei

The Features of West Nile Fever Epidemiological Situation in the World and Russia in 2013 and Prognosis of Its Development in 2014

Epidemiological situation on West Nile Fever (WNF) in Europe in 2013 was characterized by a notable rise of morbidity rate primarily due to the outbreak of WNF in Serbia (302 cases registered). In the North America, in the United States and Canada, WNF manifestations in 2013 were characterized by the lower intensity compared to previous epidemic season. 192 cases were registered in 16 constituent entities of the Russian Federation in 2013. It was revealed, that genotype 2 West Nile Virus (WNV) circulated in the territory of the Volgograd and Saratov regions, the same as in Serbia, Greece and Italy, and genotype 1 WNV in the Astrakhan region. According to the data obtained from the Reference Center for monitoring over WNV pathogen, WNV markers were detected in the territory of 61 constituent entities of the Russian Federation throughout the period of observation in 1999-2013 which testified to the existence of potential risk of human exposure during epidemic season in most of the parts of country. According to Federal Service for Hydrometeorology and Environmental Monitoring forecast, climatic conditions in Russia for the next 5-10 years will stick to global warming trend which will contribute to further spread of WNV onto the northern areas

Development of genotyping method of the glanders causative agent based on multiple locus variable-number tandem repeat analysis

The aim was to develop a short MLVA-typing scheme of the causative agent of glanders and to assess the possibility of its use for differentiation of Burkholderia mallei strains and study their genetic polymorphism.Materials and methods. The study was carried out on 14 B. mallei strains from the collection of the Volgograd Research Institute for Plague Control and 12 whole genome sequences of the B. mallei strains presented in GenBank NCBI. A set of 32 loci proposed for differentiation of the melioidosis pathogen was used to select VNTR-loci for typing the causative agent of glanders. Polyacrylamide gel electrophoresis, sequencing, and fragment analysis were applied to detect the size of the VNTR fragments.Results. VNTR loci 993, 3145, 3652, 20, 2862, and 1217, which were selected as the most variable among the causative agent of glanders, were included in the final MLVA typing scheme. The parameters of setting and detecting the results of MLVA typing have been optimized.Conclusion. Analisys of the typing results of 26 B. mallei strains showed a high discriminating power of the developed method of intraspecies differentiation of glanders pathogen based on 6-loci MLVA-scheme and the prospects of its use for epidemiological investigation to determine the source of the glanders outbreak