乌兰县鼠疫自然疫源地分离鼠疫菌株CRISPR基因分型研究  

Research on genotyping by CRISPR of Yersinia pestis isolated from the natural focus of plague in Wulan County

作　　者：张琪[1] 杨晓艳[1] 辛有全[1] 靳娟[1] 李胜[1] 柏吉祥[1] 张晓璐 李广辉 代瑞霞[1] 何建[1] ZHANG Qi;YANG Xiaoyan;XIN Youquan;JIN Juan;LI Sheng;BAI Jixiang;ZHANG Xiaolu;LI Guanghui;DAI Ruixia;HE Jian(Specialized Laboratory of Yersinia Pestis,Qinghai Institute for Endemic Disease Prevention and Control,Xining,Qinghai 810021,China)

机构地区：[1]青海省地方病预防控制所鼠疫菌专业实验室,青海西宁810021

出　　处：《医学动物防制》2024年第4期313-316,321,共5页Journal of Medical Pest Control

基　　金：国家自然科学基金项目(82260401)。

摘　　要：目的应用成簇规律间隔短回文重复序列(clustered regularly inter-spaced short palindromic repeats,CRISPR)基因分型方法,对乌兰县分离鼠疫菌株进行CRISPR基因分型分析,以了解该地区鼠疫菌CRISPR类群和基因型。方法复苏培养乌兰县1966—2010年取自鼠疫患者、媒介昆虫和喜马拉雅旱獭的63株原始鼠疫菌株,提取其DNA,设计CRISPR的YPa、YPb、YPc 3个位点引物,利用PCR技术,对以上位点进行扩增,测定其扩增产物核酸序列并进行综合分析,将测得的CRISPR序列与文献最新报道的CRISPR Dictionary和美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)数据库进行比对,探索是否存在之前未出现的CRISPR间隔(spacer)类群和基因型别,分析菌株间可能的进化关系,最终确定乌兰县喜马拉雅旱獭鼠疫自然疫源地鼠疫菌菌株的CRISPR基因库。结果63株鼠疫菌在3个CRISPR位点上共有16种spacer,其中YPa位点9种、YPb位点4种、YPc位点3种。63株鼠疫菌可被分为6个基因型,分别为G26-a1′、G26-a1′a4^(-)、G22-a4^(-)、G22、G22-a1′a4^(-)、G7,归为4大CRISPR类群,分别为Ca35′、Ca7、Ca7′、Cb4。Ca35′是该地区主要流行类群;Ca7和Ca7′分布在乌兰县东部的铜普镇和茶卡镇;Cb4仅存在于赛什克地区。结论青海省乌兰县鼠疫菌种群结构相对稳定,以Ca35′为主要种群,G26-a1′型为主要基因型,呈现显著的地区聚集特征,今后可以利用CRISPR基因分型技术加强该地区鼠疫溯源检测和防控工作。Objective This study applied the clustered regularly inter-spaced short palindromic repeats(CRISPR)genotyping method to analyze CRISPR genotyping of Yersinia pestis strains isolated in Wulan County,so as to understand the CRISPR class groups and genotypes of Y.pestis in this region.Methods Sixty-three original Y.pestis strains isolated from plague patients,vector insects and Marmota himalayana in Wulan County from 1966 to 2010 were recovered and cultured,and their DNA was extracted.Three CRISPR primers of YPa,YPb and YPc were designed,and the above loci were amplified by PCR technology.The nucleic acid sequences of the amplified products were determined and analyzed comprehensively.The CRISPR sequences were aligned with the recently reported CRISPR Dictionary and National Center for Biotechnology Information(NCBI)databases to identify unknown CRISPR spacer(spacer)class group and genotype types.Possible evolutionary relationships between strains were analyzed,and the CRISPR gene pool of Y.pestis strains in the natural focus of Marmota himalayana plague in Wulan County was finally determined.Results The 63 Y.pestis shared 16 spacers across three CRISPR loci,with 9 at YPa,4 at YPb,and 3 at YPc.The 63 Y.pestis strains could be classified into six genotypes:G26-a1′,G26-a1′a4^(-),G22-a4^(-),G22,G22-a1′a4^(-),and G7,respectively,grouped into four CRISPR groups:Ca35′,Ca7,Ca7′,and Cb4.Ca35′emerged as the main prevalent group in the region.Ca7 and Ca7′were distributed in Tongpu and Chaka towns in the east of Wulan County,while Cb4 was found only in Sashke Area.Conclusion The population structure of Yersinia pestis in Wulan County is relatively stable,with Ca35′as the main population and G26-a1′as the main genotype,showing significant regional clustering characteristics.In the future,CRISPR genotyping technology can be used to enhance the traceability detection and prevention and control of plague in this area.

关 键 词：鼠疫菌 规律成簇间隔短回文重复序列 基因分型 乌兰县 

分 类 号：R254.8[医药卫生—中医内科学]