青藏高原鼠疫自然疫源地鼠疫菌CRISPR基因分型研究

CRISPR genotyping of Yersinia pestis in the plague natural foci of Qinghai-Tibet Plateau

目的探讨青藏高原鼠疫自然疫源地鼠疫菌规律间隔成簇短回文重复序列(CRISPR)的基因型及其地区分布。方法选取青海省地方病预防控制所保存的1954-2011年在不同地区宿主、媒介体内分离的1004株鼠疫菌作为实验对象,采用传统的苯酚-氯仿混合抽提法提取鼠疫菌DNA。分别对3个CRISPR位点(YPa、YPb和YPc)进行PCR扩增、测序,将所测得CRISPR序列与文献最新报道的CRISPRDictionary数据库进行检索比对,以鉴定间区序列(spacer);对CRISPR各位点新发现的spacer,在美国国家生物技术信息中心(NCBI)数据库进行Blast序列比对,推测基因序列来源。根据CRISPR spacer阵列的多态性对青藏高原鼠疫菌进行基因分型。结果1004株鼠疫菌共发现53种spacer,其中新发现6种,分别为a105、a106、a107、b51、b52、c14;1004株鼠疫菌被分成44个不同的CRISPR基因型,10大类群,新发现基因型15种,喜马拉雅旱獭鼠疫自然疫源地鼠疫菌CRISPR基因型以G26-a1′、G7、G22、G24-a1′、G22-a1′、G9、G26-a1′a60型为主,青海田鼠鼠疫自然疫源地鼠疫菌CRISPR基因型为G37-a6′型。结论青藏高原鼠疫自然疫源地鼠疫菌CRISPR基因型具有高度多样性,且地区分布特征显著。

Objective To investigate the genotypes of clustered regularly interspaced short palindromic repeats(CRISPR)of Yersinia pestis(Y.pestis)and it's regional distribution in the plague natural foci of Qinghai-Tibet Plateau.Methods In this study,1004 strains of Y.pestis isolated from different hosts and vectors in different regions from 1954 to 2011 were selected as experimental objects,which preserved in Qinghai Institute for Endemic Disease Prevention and Control,and DNA of Y.pestis was extracted by traditional phenol-chloroform method.Three CRISPR loci(YPa,YPb and YPc)were amplified by PCR and sequenced,respectively,and the CRISPR sequences were compared with the CRISPRDictionary database recently reported in the literature to identify CRISPR spacer.For the spacer discovered for the first time at each CRISPR locus,Blast sequence alignment was performed in the National Center for Biotechnology Information(NCBI)database to speculate the source of gene sequence.Genotyping of Y.pestis from Qinghai-Tibet Plateau was performed based on the polymorphism of CRISPR spacer array.Results A total of 53 spacers were found in 1004 strains of Y.pestis,of which 6 spacers were newly discovered,namely a105,a106,a107,b51,b52 and c14.The 1004 strains of Y.pestis were divided into 10 groups with 44 different CRISPR genotypes,and 15 new genotypes were found.The dominant genotypes of Y.pestis were G26-a1',G7,G22,G24-a1',G22-a1',G9 and G26-a1'a60 in Himalayan Marmot plague natural foci,and G37-a6'in Qinghai Microtinae plague natural foci.Conclusion The CRISPR genotypes of Y.pestis in the plague natural foci of Qinghai-Tibet Plateau are high diverse and have significant regional distribution characteristics.