Genotyping Of Bacillus Anthracis By Multiple Loci Variable Number Tandem Repeats Analysis

Anthrax is a zoonotic disease caused by Bacillus anthracis, which has been used as biological weapon by terrorists to launch bio-terrorist attacks. With the development of biological weapon and the activities of international terrorist organizations, anthrax is a new developing threat to humans and animals. Therefore it is necessary to establish a rapid, effective and economical method to prevent and control outbreak of B. anthracis diseases. In this research, we analyzed the B. anthracis isolated from China by Multiple Locus Variable- Number Tandem Repeats Analysis (MLVA) in order to reveal the polymorphism of different Variable Number Tandem Repeat (VNTR) locus and the genetic relationship among major epidemic strains in China.The classic phenol chloroform extraction method was used to obtain the genome of 198 strains of B. anthracis isolated in China.18 VNTR loci were selected after biological information analysis by tandem repeats finder software. Specific fluorescent labeled primers were selected according to the information of 18 VNTR loci. The PCR products were detected by electrophoresis. The length of PCR product of all strains was analyzed by Genemarker software, and thereafter the copies of all strains were calculated. UPGMA cluster analysis method was analyzed to obtain the genetic differences among B. anthracis with the aid of bionumerics analysis software.The results showed that all B. anthracis strains were classified into 96 genotypes by UPGMA cluster analysis with 18 VNTR loci. All strains were grouped into clusters A and B, and further into group Al, A2, B1 and B2. Al group strains was divided into two subgroups A1.a and A1.b, and B1 group into two subgroups B1.a and B1.b., and B2 group into B2.a and B2.b. The main genotypes of 198 strains were mainly distributed in A1.b, B1 and B2. Al.b subgroups were mainly distributed in the sequence type 4,18 and 31, B1 group was mainly distributed in genotype 57,59,68, 77 and 78, and B2 group was mainly distributed genotype type 84 and 92. The analysis results of VNTR polymorphism information content showed that eight VNTR Locus, namely, Bams1, Bams34, A031, Bams3, VrrCl, Bams30, VrrC2 and Bams31 presented high polymorphism, thereof A031 was a novel VNTR site. Combined the eight VNTR locus with highest polymorphic information content for UPGMA cluster analysis, B. anthracis was grouped into 66 genotypes.In conclusion, the preliminary results in this thesis will promote the development of genotyping technology of Bacillus anthracis, thus ensure the level of monitoring and prevention of bio-terrorist in China.