副溶血性弧菌CRISPR的检测及其结构分析

摘要

[目的]检测副溶血性弧菌(Vibrio parahaemolyticus,简称VP)中规律成簇间隔的短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR),并对不同来源的VP中CRISPR位点的结构多样性进行分析.[方法]根据CRISPR DB数据库中公布的VP中确定的CRISPR结构序列CRISPR-1及文献中新发现的疑似CRISPR结构序列CRISPR-2设计引物,对不同来源的79株VP进行PCR扩增.利用CRISPR Finder分析CRISPR结构,采用生物信息学方法对不同来源VP的CRISPR位点结构多样性进行比较分析.[结果]79株VP中CRISPR-1的检出率为92.41％,CRISPR-2的检出率为96.20％,同时具有这2个位点的菌株占总数的89.87％,只有1株菌被检出不含有任何位点.分别比较不同来源的菌株CRISPR-1、CRISPR-2位点的重复序列发现不存在序列差异,而临床菌株的这2个CRISPR位点在间隔序列上比环境分离菌株存在更多的变异.2个CRISPR位点根据间隔序列的不同在VP中一共组成8种CRISPR谱型(编号A-H),除F谱型外,A-E、G谱型均只在临床分离菌株中发现,而在环境分离菌中还发现不含任何位点的H型.[结论]CRISPR在VP中普遍存在.环境分离菌株与临床分离菌株中CRISPR的结构存在差异.%[Objective] To detect the clustered regularly interspaced short palindromic repeats (CRISPR) in Vibrio parahaemolyticus,and analyze its structural diversities in different sources of V.parahaemolyticus strains.[Methods] The primers of convincing CRISPR structure CRISPR-1 were designed by using the sequence according to CRISPR database,while the primers of questionable CRISPR structure CRISPR-2 were designed according to the literature.PCR approach was used to detect the CRISPR locus in all 79 strains,and all CRISPR sequences were analyzed using CRISPR Finder.Furthermore,the structural diversities of CRISPR in different sources of V.parahaemolyticus were analyzed using bioinformatics methods.[Results] The positive rate of CRISPR-1 was 92.41％ and of CRISPR-2 96.20％.The strains possessed with these two locus accounted for 89.87％ of the total strains,and only one strain did not contain any locus.There was no difference between the repeats of CRISPR-1 and CRISPR-2 from different sources of V.parahaemolyticus,whereas the spacers of those two locus in clinical strains had more variations than those in environmental strains.Therefore,two CRISPR locus formed 8 spectral patterns (numbers A-H) according to the variations of spacers.Except type F,the patterns A-E,G only were found in the clinical strains.And the type H containing none of locus was only found in environmental strains.[Conclusion] CRISPR commonly existed in V.parahaemolyticus.There were differences in the structure of CRISPR between environmental and clinical strains.