1954—2011年青海高原鼠疫耶尔森菌CRISPR基因分型及地区分布

Genotyping by CRISPR and regional distribution of Yersinia pestis in Qinghai-plateau from 1954 to 2011

目的 研究青海高原鼠疫耶尔森菌（鼠疫菌）的CRISPR基因型（类群）及其地区分布.方法 选取1954—2011年间青海高原境内各县/市从人鼠疫患者、宿主动物及媒介昆虫体内分离的鼠疫菌102株,采用传统的十二烷基磺酸钠裂解和苯酚-氯仿法提取鼠疫菌DNA.分别对YPa、YPb和YPc等3个CRISPR位点进行PCR扩增、测序,然后将所测得CRISPR序列与文献最新报道的CRISPR Dictionary和NCBI数据库检索比对,以鉴定CRISPR spacer阵列.最后根据CRISPR spacer阵列的多态性对青海高原鼠疫菌进行基因分型,并描绘其地区分布特征.结果 102株鼠疫菌共发现40种spacer,包括YPa 22种、YPb 13种、YPc 5种,其中有5种新spacer,分别为a1＇、a103、a104、b4＇和b4＇;Ypa、Ypb和Ypc分别鉴定出16、10和5种不同的spacer阵列,新发现的spacer阵列为11种.102株鼠疫菌可被分为24个基因型,共归为9大CRISPR类群,分别为Cb4、Cb4＇、Cb2、Ca37、Ca7、Ca7＇、CaΔ5＇、Ca35＇和Cc3＇,其中Ca7、Ca7＇、CaΔ5＇、Ca35＇呈现出显著的聚集性特征：Ca7主要分布在玉树、囊谦、称多、杂多、治多和曲麻莱;Ca7＇分布于循化、同仁、泽库、同德、玛沁和贵南;CaΔ5＇集中在祁连、刚察、门源、大通;Ca35＇分布在湟源、海晏、刚察、天峻、德令哈、乌兰、都兰、兴海、共和和贵德.结论 青海高原鼠疫菌CRISPR种群结构复杂,Ca7、Ca7＇、CaΔ5＇和Ca35＇是流行最普遍的主要种群,这些种群呈现显著的地区分布特征,可以利用CRISPR分型技术加强该地区鼠疫检测和防控工作.

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.