霍乱弧菌基因表达分析中内参基因的选择  

Selection of reference genes for gene expression analysis in Vibrio cholerae

作　　者：张翠彩[1] 逄波[1] 蒋秀高[1] 阚飙[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206

出　　处：《中国人兽共患病学报》2014年第5期433-438,共6页Chinese Journal of Zoonoses

基　　金：国家科技重大专项(No.2012ZX10004-215);国家自然科学基金(No.30800987);传染病预防控制国家重点实验室资助项目(No.2012SKLID203)~~

摘　　要：目的确定不同实验条件下基因表达分析中的稳定内参基因。方法以霍乱弧菌O1群El Tor菌株为研究对象,利用qRT-PCR方法比较不同pH值(pH 8.0、pH 5.5)以及不同温度(37℃、30℃)等多种生理、外界环境模拟培养条件下,thyA、recA、rpoA、gyrB、16SrRNA以及VCA0862 6个候选内参基因的表达水平,利用geNorm软件评价其稳定性。结果不同培养条件下,6个候选内参基因表达水平存在差异。不同pH值条件下最佳基因是recA;不同温度条件下最佳基因是gyrB;不同pH值及温度综合评价时,最佳基因是recA。结论本研究评价和提出了细菌基因表达分析中内参基因筛选的重要性,不同实验条件下最稳定内参基因是不同的,针对不同条件下的基因转录定量分析,应分别对内参基因稳定性进行评价并选择最稳定基因。In order to select good reference genes for normalization,a set of most commonly used reference genes for reliable gene expression analysis in Vibrio cholerae was explored in this study.A panel of physiological and environment imitateculturing conditions were performed including different pH values （pH 8.0 and pH 5.5） and different temperatures （37 ℃ and 30 ℃）.The qRT-PCR was used to detect the gene expression level of six reference genes （thyA,recA,rpoA,gyrB,16SrRNA,and VCA0862） in Vibrio cholerae O1 El Tor strain.The gene expression stabilities of the six reference genes were evaluated using the freely distributed Microsoft Excel application geNorm.The results showed that the gene expression level of six candidate reference genes was different under different culture conditions.The recA and gyrB turned out to be the most suitable reference genes under different pH values and different temperatures,respectively.While recA turned out to be the most suitable reference gene under both different pH values and temperatures.In this work,we evaluated and stated the importance of selecting the reference genes for quantitative gene expression analysis by qRT-PCR in bacteriology.The results demonstrated that the most stable reference gene is different for different conditions respectively.It may be needed to revalue the gene expression stability of candidate reference genes as internal controls when the conditions are changed.

关 键 词：霍乱弧菌 QRT-PCR 内参基因 GENORM 

分 类 号：R378[医药卫生—病原生物学]