青海省格尔木市鼠疫耶尔森菌生化特征及基因分型研究

Biochemical characteristics and genotyping of Yersinia pestis in Golmud, Qinghai

目的 了解青海省格尔木市鼠疫耶尔森菌(Yersinia pestis)的生化特征,进行规律成簇的间隔短回文重复序列(CRISPR)基因分型和差异区段(DFR)研究,为青海省格尔木市鼠疫菌鉴定溯源、鼠疫科学防控提供理论依据。方法 选取1967年以来青海省格尔木市境内从人尸、宿主动物及媒介昆虫体内分离出28株鼠疫菌,通过糖醇酵解实验,研究其生化特征。采用十二烷基磺酸钠裂解和苯酚-氯仿法提取鼠疫菌DNA。分别对CRISPR的YPa、YPb和YPc3个位点进行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省格尔木市鼠疫菌进行基因分型。采用23对DFR(DFR01-DFR23)分型引物和质粒验证引物(PMT1)对试验菌株DNA进行分型验证。结果 鼠疫菌糖醇类酵解表型特征研究结果显示,28株鼠疫菌对甘油、阿胶糖、麦芽糖的酵解均为阳性,对鼠李糖、蜜二糖、脱氮的酵解均为阴性。CRISPR分型发现10种spacer,包括YPa 5种、YPb 4种、YPc 1种,a105和b51是新发现spacer。28株鼠疫菌归类为8个基因型,分为5个CRISPR类群:Cb4、Cb4’、Ca7、Ca7’和Ca35’。DFR分型可分为4个基因组型,分别为32型、5型、8型和49型。该地区主要基因组型为32型和5型。结论 青海省格尔木市鼠疫菌均为青藏高原型,当地鼠疫菌基因型较少,遗传较为稳定,具有明显的地区分布特征。同一地区不同菌株基因型有差异,存在鼠疫菌菌株微进化。

Objective To understand the biochemical characteristics of Yersinia pestis in Golmud City,Qinghai Province,and conduct clustered regularly interspaced short palindromic repeats(CRISPR) and different region genotyping(DFR) to provide scientific basis for Yersinia pestis identification,traceability,plague detection and control in Golmud City,Qinghai Province.Methods Twenty-eight isolates of Y.pestis were isolated from human plague patients,host animal and vectors insect in Golmud City,Qinghai Province since 1967.And their biochemical characteristics were studied by glycolysis experiment.Y.pestis DNA was extracted by sodium dodecyl sulfonate lysis and phenol-chloroform method.The three CRISPR loci of YPa,YPb and YPc were amplified and sequenced by PCR respectively,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 geno typing of isolates of Y.pesits were finally conducted according to the polymorphism of the spacer arrays and the regional distribution pattern of isolates of in Y.pestis in Golmud City.Twenty-three pairs of DFR(DFR01-DFR23) typing primers and plasmid validation primers(PMT1) were used for DNA typing verification.Results The results showed that the 28 strains were positive to glycerol,donatose and maltose,but negative to rhamnose,miraeosaccharide and denitrification.The 28 strains of Y.pestis were all of the Qinghai-Tibet Plateau type.Ten spacers including 5 of YPa,4 of YPb and 1 of YPc were observed among 28 isolates of Y.pestis in Golmud,of which 2 spacers(a105 and b51’ were firstly identified.Twenty-eight isolates were divided into 8 CRISPR genotypes and classified into 5 CRISPR clusters(Cb4,Cb4’,Ca7,Ca7’ and Ca35’).4 genotypes were found in DFR study,which were genomovar 32(16 strains),genomovar 5(9 strains),genomovar 8(2 strains) and genomovar 49(1 strain).Genomovar 32 and genomovar 5 were the main genotypes in this region.Conclusions All the Y.pestis strains in Golmud City of Qinghai Province are of Qinghai-Tibet Plateau type.The local Y.pestis genotypes are relatively stable and has obvious regional distribution characteristics.The genotypes of different strains in the same area are different,and there is microevolution of Y.pestis strains.