| Objective: To analyze the polymorphism and evolutionary characteristics of the different strains of Brucella in our country in different times and places strain separated from our country, to develop the proper taxonomic method and explore the research on the type of Brucella. In order to investigate genetic relationship within the group and identify potential diagnostic markers we have, to provide database on the molecular biological research on the molecular epidemic of Brucellosis.Methods: The multiple locus sequence typing technology (MLST) was used to sequence 7 house keep genes, 1 outer membrane gene and 2 Virulence factors from T4SS. The sequences are compared to that of the different alleles of these genes to determine the sequence types. Using online software to analysis the allele sequence,linkage equilibrium,clustering,phylogenetic trees and so on, to understand the evolutionary relationship between strains.Results:(1) In study,17 strains of the international standards Brucella strains as a reference,the result show differences for 63/9(cobQ),86/8/59(glk),Tulya(aroA),5K33(omp25),Complete accuracy of strains is 81.25%,The accuracy of alleles is 96.88%。(2) Selectde 164 stains isolated from China from 1953 to 2010. Brucella melitensis,bv1,bv2,bv3 respectively 38,21,36 strains;Brucella abortus,bv1,bv2,bv3,bv6,bv7,bv9 respectively 13,1,19,2,1,5 strains;Brucella suis, bv1,bv3 respectively 16,6 strains;Brucella canis is 16 strains. After serological methods,phage rechecked,are consistent with the original results.(3) Analysis of alleles showed omp25 polymorphisms variation degree is the largest. Gap,aroA,glk,dnaK,gyrB,trpE,cobQ,omp25,VirB5,VirB8 the genetic polymorphisms is 0.6612,0.7867,0.7286,0.5663,0.7035,0.4110,0.7114,0.8996,0.6659,0.5780.(4) Concatenated sequence data were used to construct an unrooted neighbour-joining tree representing the relationships between STs. This shows that four previously characterized classical Brucella species to well-separated clusters. B. canis isolates are located on the same branch very closely related to B.suis. found same species-specific markers.Conclusion:(1) The sequence database provides a powerful dataset for addressing ongoing controversies in Brucella taxonomy. Furthermore, by using the phylogenetic backbone described here, robust and rationally selected markers for use in diagnostic assay development can be identified. MLST typing of Brucella can be used as an important means of evolutionary relationships. (2) Compared to the biological type and the ST type, fonud no correlation between of them. MLST further confirmed Brucella canis is not just a biological type.(3) Find some specific SNPs: Brucella abortus (except Tulya) as gap-2;Brucella melitensis including three SNPs,that is glk-3,trpE-5,VirB5-4; Brucella ovis also including three SNPs,that is aroA-3,VirB5-7,VirB8-5;dnaK-1 for most Brucella suis and Brucella canis sharing (except 513 ).(4) In this study Used of 10 loci, based on the DNA sequence typing, separation of the unknown Strain identification of species is a useful tool. |
PDF Full Text Request