成簇的规律间隔短回文重复序列技术在鼠疫菌基因分型中的应用  

Application of clustered regularly interspaced short palindromic repeats in genotyping of Yersinia pestis

作　　者：王海峰[1] 陈永明[1] 周松[1] 牛艳芬[1] 杨晓燕[1] 张懿晖[1] 刘广[1] 杜国义[1] 刘合智[1] 史献明[1] WANG Hai-feng;CHEN Yong-ming;ZHOU Song;NIU Yan-fen;YANG Xiao-yan;ZHANG Yi-hui;LIU Guang;DU Guo-yi;LIU He-zhi;SHI Xian-ming(Department of Laboratory,Anti-plague Institute of Hebei Province,Zhangjiakou,Hebei 075000,China)

机构地区：[1]河北省鼠疫防治所检验科,河北张家口075000

出　　处：《中国媒介生物学及控制杂志》2021年第1期30-33,共4页Chinese Journal of Vector Biology and Control

基　　金：河北省2017年度医学科学研究重点课题计划(20170466);河北省2020年度医学科学研究课题计划(20200025);国家科技重大专项(2018ZX10713-001-002)。

摘　　要：目的针对分离自内蒙古高原长爪沙鼠鼠疫自然疫源地的鼠疫耶尔森菌(鼠疫菌),进行DNA成簇的规律间隔短回文重复序列(CRISPR)分型,对比同一类型疫源地,不同地区分离的菌株基因型,建立沙鼠鼠疫疫源地鼠疫菌株的CRISPR基因库,为疫情的追溯和流行病学分析奠定基础。方法应用3对CRISPR引物(YPa、YPb、YPc)将实验菌株DNA进行聚合酶链式反应(PCR)扩增并测序,然后将所测得CRISPR序列与文献最新报道的CRISPR Dictionary比对,找出CRISPR spacer阵列,确定基因型别,最后用Bionumerics 7.6软件制作聚类图,分析其进化关系。结果33株鼠疫菌共发现9种spacer,包括4种YPa(a1、a2、a3、a56)、2种YPb(b1、b2)和3种YPc(c1、c2、c3),经比对所有菌株被归为1个CRISPR基因簇(Cb2),2个基因型,内蒙古自治区(内蒙古)鄂托克旗和杭锦后旗菌株为1个新基因型,命名为2′型(a1-a2-a3-a56,b1-b2,c1-c2-c3),内蒙古乌拉特前旗、河北省康保县和宁夏回族自治区银川市的菌株均为基因1型(a1-a2-a3,b1-b2,c1-c2-c3)。结论鼠疫菌在内蒙古长爪沙鼠鼠疫疫源地整体上遗传稳定,归于1个基因簇,但是也存在一些微进化,体现在不同地区的菌株有不同的基因型。Objective To investigate the genotype of Yersinia pestis isolated from the natural plague focus of Meriones unguiculatus in Inner Mongolian Plateau using clustered regularly interspaced short palindromic repeats(CRISPR)and the difference in the genotype of strains from the same type of epidemic focus and different areas,to establish the CRISPR gene bank of plague strains from gerbil plague foci,and to lay a foundation for epidemiological trace-back and analysis of epidemic situation.Methods Three pairs of CRISPR primers(YPa,YPb,and YPc)were used for PCR amplification and sequencing of the DNA of experimental strains,and the CRISPR sequence obtained was compared with the latest CRISPR Dictionary reported in literature to obtain CRISPR spacer array and identify genotype.Bionumerics 7.6 software was used to plot clustering charts and analyze the phylogenetic relationship.Results A total of 9 spacers were found in 33 strains of Y.pestis,i.e.,4 types of YPa(a1,a2,a3,and a56),2 types of YPb(b1 and b2),and 3 types of YPc(c1,c2,and c3).The comparative analysis showed that all strains were classified as 1 CRISPR gene cluster(Cb2)with two genotypes.The strains isolated from Etuoke banner and Hanggin Rear banner of Inner Mongolia were identified as a new genotype,which was named as genotype 2′(a1-a2-a3-a56,b1-b2,c1-c2-c3),and the strains from Urad Front banner of Inner Mongolia,Kangbao county of Hebei province,and Yinchuan city of Ningxia Hui autonomous region were identified as genotype 1(a1-a2-a3,b1-b2,c1-c2-c3).Conclusion Y.pestis isolated from the plague foci of M.unguiculatus in Inner Mongolian is genetically stable as a whole and is classified as one gene cluster,but a certain degree of microevolution is observed,which reflects that strains from different areas have different genotypes.

关 键 词：鼠疫耶尔森菌 成簇的规律间隔短回文重复序列 基因分型 

分 类 号：Q78[生物学—分子生物学] R378.61[医药卫生—病原生物学]