Affiliation:
1. Federal Center for Toxicological, Radiation and Biological Safety
Abstract
The purpose of the study is to evaluate the effectiveness of using the developed MLVA protocol for differentiating strains of the causative agent of brucellosis. This protocol includes the analysis of 15 VNTR loci using modified MLVA primers. For in vitro testing of the proposed MLVA scheme, we used previously isolated DNA from strains B. canis RM 6/66, B. suis 1330, B. suis 183-L, B melitensis 1565. MLVA was carried out by PCR followed by separation of amplicons in an agarose gel. Positive amplification was observed for 10 of the 15 VNTR loci, namely Bru6, Bru7, Bru9, Bru16 and Bru18, Bru19, Bru21, Bru30, Bru43 and Bru45. The molecular size of these loci for the reference strains B. canis RM 6/66 and B. suis 1330 was confirmed in silico. MLVA results for strains represented in the GenBank database are also presented. By searching the NCBI resource databases, we obtained the genomic sequences of 49 Brucella strains of the species B. canis, B. suis, B. aborus, and B melitensis. Using bioinformatic analysis, the molecular weight of each of the ten VNTR loci and the number of repeats in it were determined for these strains. Based on the results of the MLVA, a dendrogram was constructed. Based on a phylogenetic ana¬lysis of the sequences of ten variable loci, it was established that the majority of the studied Brucella strains were distributed on the dendrogram in accordance with their taxonomic position. Thus, we concluded that our proposed MLVA protocol has the potential to be used for the differentiation of Brucella strains.
Publisher
Krasnoyarsk State Agrarian University
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