Genotyping Of Yersinia Pestis By Multiple Locus Variable Number Tandem Repeat And Different Region And Its Epidemiological Significance In Yunnan

[Objective]To identify genotypes of Yersinia pestis in Yunnan, we have used multiple locus variablenumber tandem repeat analysis(MLVA) and different region(DFR). It can explored theintrinsic relationship among different ecotypes,.It can also provide evidence for theprevention and control of plague.[Methods]Methods of MLVA:1. Selected strains We have selected158strains of Yersinia pestis of Yunnan whichcome from different areas, different times and different source. They contain139strains of commensal rodents,16strains of wild rodents,7stains of Yulong.Besides, there is1strain of EV.2. Selected VNTR14VNTR sites have been selected which are based on the13strains published whole genome alignment.12of them are new sites and2ofthem have been used.3. DNA extraction The QIAGEN bacterial DNA kit has been used to extractYersinia pestis.4. PCR and capillary electrophoresis Using25l of multiplex PCR amplification,the products are analyzed by capillary electrophoresis, the number of repeat unitsare calculated by PCR product size, the final results are saved in Excel format.5. Analysis results The data are analyzed by BioNumerics software in form ofclustering maps and the minimum spanning tree.Methods of DFR:1. Selected strains We have selected171strains of Yersinia pestis of Yunnan whichcome from different areas,different times and different source. They contain148strains of commensal rodents,16strains of wild rodents,7stains of Yulong. 2. Selected DFR23DFR are selected according to the relevant paper.3. DNA extraction The QIAGEN bacterial DNA kit has been used to extractYersinia pestis.4. PCR and agarose gel electrophoresis Using25l of multiplex PCR amplification,the products are subjected to agarose gel electrophoresis and analyzed by gelimager, the results are recorded as “+”、“－” to save the Excel format.5. Results The data are analyzed by BioNumerics software in form of clusteringmaps and the minimum spanning tree.[Results]Results of MLVA:1. The resolution of14sites are largely different,8sites that the Simpson’s index ofdiversity are0, the others that M52、M59、MLV7、MLV8、MLV4have certainresolution.2. The clustering of genotype showed that these strains could be classified into2groups,3clusters and5genotypes.3. The wild rodent strains belonged to cluster A, the strains isolated in Yulongbelonged to cluster B and commensal rodent strains belonged to cluster C. Theresults were consistent with traditional ecological classification. The wild rodentstrains, commensal rodent strains and Yulong strains isolated in Yunnan belongedto different gene clusters, but Yulong strains and wild rodent strains were in onegroup.Results of DFR:1. A total of171Yersinia pestis are divided into7genotypes by23DFRs, which areGenomovar5, Genomovar7, Genomovar9and4newly discovered genotypescalled Genomovar-yn1、yn2、yn3、yn4.2. All strains of Yulong are Genomovar5.16strains of wild rodents are divided into3genotypes,13of them are Genomovar7,2of them are Genomovar9, one ofthem is Genomovar-yn1.148strains of commensal rodents are divided into4genotypes,145of them are Genomovar9,3of them are respectivelyGenomovar-yn2、-yn3、-yn4.3. The similarity of genotype between Yulong plague and highland of northwesternYunnan Province type (wild mouse plague) is high that87.2%while the similaritybetween Yulong plague and the residential area of Yunnan and Fujian Provinces type(house mouse plague) is low that73.75%.[Conclusion]1. On this study, we can choose5VNTR sites and23DFR to genotype the Yersiniapestis in Yunnan and build the rapid identification traceable.2. The MLVA type、 DFR type of Yulong strains are largely different from thecommensal rodents and wild rodents in Yunnan, it is low similar with commensalrodents that the residential area of Yunnan and Fujian Provinces type, it is highsimilar with wild rodents that the highland of northwestern Yunnan Province type.Combined with biochemical characteristics, it is suggested that Yulong plague fociis a new foci.3. The results of clustering showed that two genotyping results is agreement withecological of Yunan.4. On this study, the results of MLVA、DFR have enriched the database of Yersiniapestis, provided the foundation for future studies and provided strong basis for theprevention and control of plague.