Characterization Of Yersinia Pestis Isolates Dispersed Across China By Multiple-Locus Variable-Number Tandem Repeat Analysis

Varible number of tandem repeat(VNTR) is some DNA sequence repeated side by side,which occurs in large quantities in eukaryotic and prokaryote,consists of simple homopolymeric tracts of a single nucleotide type or of large or small numbers of several multimeric.These sequence elements showed hypervariablity among individuals because the copy numbers of the simple sequence motif differ in individuals.Recently,it has been applied to molecular epidemic investigation, identification and genotyping of strains.The chromosome of Yersinia pestis is 4.59～4.66Mb in length,among which there are 4037～4198 open reading frame(ORF) and 47.6～50%GC contents.Typical Y.pestis strains contain three plasmids pPCP1,pCD1 and pMT1.The pPCP1 and pMT1 plasmid is special for Y.pestis,which encode associated pathogenic genes and virulence factors.The analysis of Y.pestis genomic sequence shows that lots of repeat arrays are randomly distributed in the Y.pestis genome.Diversity exists in the same VNTR locus between different strains.According to this fecture,we selected sixteen VNTR loci for analysis in 213 isolates dispersed across China(belong to 11 natural foci and 18 ecotypes) and EV strain in the study.We obtained the repeat units by PCR amplification,agrose-gel electrophoresis,molecular mass calculation, sequencing and sequence intercomparison.Then we analyzed data by BioNumerics software.According to the results of sequence intercomparison,we discovered that among the sixteen loci,mutations exist in core sequence of some loci.There are certain regularities,that is the position of mutation is fixed,only the base transition and transversion occur but not insertion and deletion.Besides,the copy number of Y. pestis strains in China varied discontinuously.And in some loci,there is only one strain which have certain copy number.(There is no copy number fifteen in all loci.)We studied optimum association of VNTR loci by analysis of polymorphism index of sixteen loci.Then we determined ten loci which can be used in conducting molecular epidemiological source-tracking.The results of MLVA based on ten loci showed that 214 Y.pestis isolates are divided into 14 clades and 74 MLVA genotypes,which confirm the diversity and stability of Y.pestis isolates in China in genetic evolution process.The results also showed that MLVA genotypes agree well with ecotypes of Y.pestis isolates in China.So we constructed organic connection between MLVA genotypes and ecotypes,and MLVA genotypes further supplement and enrich the ecotypes.While the result of Minimum spanning tree suggested evolution relationship of different plague natural foci.MLVA and field epidemiological investigation were first applied in dealing with sudden epidemic situation of plague in Yulong,YunNan.The result showed that the copy number of 16 loci are completely identical among five Y.pestis isolates in Yulong,YunNan.They belong to the some MLVA genotype,which suggested they were from the same epidemic process.However the result of cluster analysis showed that genetic relationship of strains in Yulong and Tibetan Platue are closer, which provides scientific basis for further determining the property of plague nature foci and the infection source.