青海省海西州鼠疫自然疫源地分离鼠疫菌株CRISPR基因分型研究

CRISPR genotyping analysis of Yersinia pestis in Haixi,Qinghai Province

目的利用DNA规律聚集簇间隔短回文重复序列(Clustered regularlyinter-spaced short palindromic repeats CRISPR)分型方法,全面探索海西地区分离鼠疫菌株是否存在新的基因型,为鼠疫菌溯源鉴定及流行病学分析提供理论依据。方法分离培养海西地区1961-2009年间取自鼠疫患者、媒介昆虫及中间宿主的50株鼠疫菌后提取其DNA,利用PCR技术,对鼠疫菌CRISPR分型中的YPa、YPb、YPc 3个位点进行扩增,测定其扩增产物核酸序列并进行分析,将测得CRISPR序列与文献最新报道的CRISPR Dictionary和NCBI数据库进行比对,找出新的CRISPR spacer阵列,基因型别,分析其进化关系,最终确定青海省海西州喜马拉雅旱獭鼠疫自然疫源地鼠疫菌株的CRISPR基因库。结果50株鼠疫菌在3个CRISPR位点上共有24种spacer,其中YPa位点上有13种、YPb位点8种、YPc位点3种,b51是新发现的spacer。50株鼠疫菌可被分为16个基因型,共归为6大CRISPR类群。Ca35′是该地区主要流行类群;Ca7是流行最为广泛的类群;Cb4和Cb4′仅存在于格尔木唐古拉地区。结论青海省海西州地区鼠疫菌种群结构复杂,Ca35′、Ca7、Cb4′是流行最普遍的种群,G26-a1′型、G22型、G9型、G26-a1′a4-为主要基因型,呈现显著的地区分布特征,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。

Using a DNA clustered regularly inter-spaced short palindromic repeats typing method,we comprehensively explored the existence of new genotypes of isolated plague strains in Haixi,to provide a theoretical basis for the traceability identification and epidemiological analysis of Y.pestis.The DNA of 50 Y.pestis strains was isolated from patients with plague,insectvectors,and intermediate hosts between 1961 and 2009.PCR was used to amplify YPa,Ypb,and YPc in Y.pestis CRISPR typing.The nucleic acid sequences of the amplified products were determined and analyzed,and the detected CRISPR sequences were aligned to the CRISPR dictionary and NCBI databases recently reported in the literature.New CRISPR spacer arrays and genotypes were determined,evolutionary relationships were analyzed,and the CRISPR gene pool of the plague strain in Haixi,Qinghai Province,was finally determined.The 50 Y.pestis strains shared 24 spacers at three CRISPR sites,including 13 at YPa sites,8 at YPb sites,and 3 at YPc sites;b51 was a newly discovered spacer.The 50 strains were classified into 16 genotypes,grouped into six CRISPR groups.Ca35'was the main prevalent group;Ca7 was the most widely prevalent taxon;and Cb4 and Cb4'were present only in Golmud.The structure of Y.pestis species in Haixi,Qinghai Province,is complex.Ca35',Ca7,and Cb4'were the most prevalent populations.G26-a1',G22,G9,and G26-a1'a4 were the main genotypes,and showed substantially different regional distribution characteristics.In the future,CRISPR typing technology may be used to strengthen source detection,and prevention and control measures for plague in this region.