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Objective@#To investigate the CRISPR genotypes (clusters) and regional distribution of Yersinia pestis in Qinghai-plateau.@*Methods@#One hundred and two isolates of Y. pestis isolated from human plague patients, host animal and insect vectors from Qinghai-plateau were selected. The DNAs were extracted using the traditional sodium dodecyl sulfate decomposition and phenol-chloroform method. Three CRISPR loci YPa, YPb and YPc of 102 isolates of Y. pesits were amplified and sequenced, and then the CRISPR sequence analysis was carried out by comparing the latest published CRISPR spacer dictionary and the NCBI database to identify the spacer and spacer array. CRISPR genotyping of isolates of Y. pesits were finally conducted according to the polymorphism of the spacer arrays and the regional distribution pattern of isolates of Y. pesits in Qinghai-plateau was described.@*Results@#Forty spacers including 22 of YPa, 13 of YPb and 5 of YPc were observed among 102 isolates of Y. pestis in Qinghai-plateau, of which 5 spacers (a1', a103, a104, b4'' and b4''') were firstly identified. Meanwhile, 16, 10, and 5 different spacer arrays were obtained in YPa, YPb and YPc respectively, including 11 new spacer arrays detected in this study. One hundred and two isolates were divided into 24 CRISPR genotypes and classified into 9 CRISPR clusters (Cb4, Cb4', Cb2, Ca37, Ca7, Ca7', CaΔ5', Ca35' and Cc3'). Each dominant cluster presented significant aggregation geographically: Ca7 were found in Yushu, Nangqian, Chenduo, Zaduo, Zhiduo and Qumalai countries. Ca7' were found in Xunhua, Tongren, Zeku, Tongde, Maqin and Guinan countries. CaΔ5' were restricted to Qilian, Gangcha, Menyuan and Datong countries. CaΔ35' were found in Huangyuan, Haiyan, Gangcha, Tianjun, Delingha, Wulan, Doulan, Gonghe, Xinghai, Guide and Tongde countries.@*Conclusion@#CRISPR-based genotyping analyses showed complicated population of Y. pestis in Qinghai-plateau. Four clusters, Ca7, Ca7', CaΔ5' and Ca35' were the most epidemic dominant four clusters and presented obvious regional distribution patterns, which instructed us to strengthen the surveillance and prevention and control by CRISPR-genotyping technique.
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Objective To construct a technical platform for scarless gene modification of Yersinia pestis and to study the functions of its specific genes.Methods The resistance fragment, including upstream and downstream homologous arms of targeted regions, was reamplified by asymmetric PCR.The amplicons were introduced into Y.pestis harboring plasmid pKD46.With the induction of L-arabinose,the recombinant related enzymes: Exo, Beta and Gam, were expressed to guide the homologous recombination.A donor plasmid, pKSI-1, which carried the desired modification fragment flanking by I-SceⅠ recognition sites, was introduced into Y.pestis as the second step of λ-Red recombination with the help of pREDTKI.Results and Conclusion Two mutant strains:△waaA and waaA(△9nt), were successfully constructed for Y.pestis strain 201.Scarless modification introduces no extra modification to the genome, and it is ideal for comprehensive functional genomic studies.
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Objective To investigate the biological characteristics and epidemiological significance of Yersinia pestis strains in Qilian County,Qinghai Province,in order to provide a scientific basis for plague prevention and control.Method Totally 67 strains were separated from kinds of host in Qilian County,Qinghai Province from 1958 to 2011,to do biochemical test,toxicity test,virulence factors evaluation,plasmid analysis and different region (DFR) genotyping.Results According to biochemical typing,48 of the 50 strains tested were Qing-Tibet Plateau ecotype,15 were Qilian Mountain ecotype,and the remaining 4 were different ecotypes from the plague foci in Qinghai plateau.The strains had 8 genomovars,and were given priority to genomovar8 (42 strains),secondly,genomovar44 (15 strains),genomovar5 (4 strains),genomovar7 (2 strains),genomovar19 (1 strain),genomovar30 (1 strain),genomovar32 (1 strain),and genomovar34 (1 strain).A proportion of 95.52％ (64/67) of the strains contained 3 kinds of plasmid-6 × 106,45 × 106,and 52 × 106;85.07％ (57/67) contained all the four virulence factors,and 96.00％ (48/50) were velogenic strains.Conclusion The strains separated in Qilian County,Qinghai Province have the characteristics of Qinghai-Tibet Plateau plague's pathogen and have strong toxicity,so we should enhance the plague monitoring and give more publicity to plague prevention to prevent animal plague spreading to human.
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Objective By the method of multiple polymerase chain reaction (PCR),we intend to amplify different regions (DFR) of Yersinia pestis DNA,and to establish a multiple DFR genotyping technique for detection of Yersinia pestis.Methods According to the product size of 23 DFRs and pMT plasmid,24 primers were optimized and combined,then multiple primers in one PCR reaction system were added,and positive template DNA was amplified.Meanwhile,200 wild strain DNAs were amplified by multiple PCR and normal PCR,to verify the coincidence rate of the two methods.Results Totally 24 target segments were amplified through the positive DNA template.Through different permutation and combination,24 primers were optimized and combined into 9 groups.Totally 200 wild strain DNAs were used for verification,the coincidence rate of multiple PCR and normal PCR was 100％.Conclusions Multiple PCR is applicable and feasible for DFR genotyping of Yersinia pestis.It is an efficient,economic and high accuracy experimental method for large quantities of Yersinia pestis DFR genotyping.
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Bacteriophages are abundantly distributed viruses , which are able to infect bacteria .Bacteriophages are becoming a focus of attention for microbiologists , as they can cause pollution to the fermentation industry and might serve as alternative therapies for antibiotic-resistant pathogenic bacteria .Effective bacteriophage infection normally involves adsorp-tion, injection, replication, assembly and release , against which bacteria have developed various resistance strategies .The research progress in the bacteriophage resistance mechanisms is briefly reviewed in this paper .
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Objective To investigate the type of plasmid map of Y. pestis in the R. opimas natural plague loci in Junggar Basin. Methods A total of 39 plasmid DNA of Y. pestis which were isolated from the natural plague loci of Junggar Basin, Tianshan Mountain, Kunlun Mountain, Qinghai-Tibet Plateau and In-ner Mongolia were extracted by the methods of Kado and Liu. The plasmid map was analyzed by the methods of agarose gel eleetrophoretogram. Results Two types of plasmid map were found in 26 Y. pestis which were isolated from Junggar Basin. Of them 23 were 6 × 106, 45 × 106 and 65 × 106 type of plasmid map, and 3 were 6 × 106, 45 × 106 and 72 × 106 type. Conclusion There are two types of plasmid map in the R. opi-mus natural plague loci in Junggar Basin. One type, which is the dominant type in this area, is 6 × 106, 45 × 106 and 65 × 106 type. This type is also similar to the dominant plasmid map type of the nature plague loci of Tianshan Mountain, Kunlun Mountain, Qinghai-Tibet Plateau and Inner Mongolia. The other type is 6 × 106, 45 × 106 and 72 × 106 type, and this type is new plasmid map type of Y. pestis in our country.
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Objective To purify native F1 antigen from E pestis EV76 strain and determine its ef-ficacy against Y. pestis. Methods A new purification method was developed by the substitution of physical disruption ( glass beads) for organic solvent ( acetone and toluene) one, followed by a combination of ammo-nium sulfate fractionation and SephacrylS-200HR column filtration chromatography. Groups of mice were im-munized with F1 antigen adsorbed to 25% aluminum hydroxide in PBS by intramuscular route. The immu-nized animals were challenged subeutaneously(s, c. ) with 104 CFU of Y. pestis strain 141 at 18 weeks after the primary immunization. Results There was no IgG titre difference between two groups of mice with one-dose immunization, whereas in the two-dose immunization groups, the group F1-40 μg induced a statistically higher antibody titre than the group F1-20 μg. Complete protection was observed for animals immunized with purified F1 antigen by s.c. route. In contrast, the control mice immunized with aluminum hydroxide suc-cumbed to a same dose of Y. pestis 141 challenge. Conclusion This purification strategy is a simple and ef-fective, and can be operated in a large scale. Native F1 antigen extracted from Y. pestis EV76 is highly im-munagenic, and can be used as a key antigen component to develop sub-unit vaccine of plague.