维氏气单胞菌双基因PCR检测方法的建立  

Establishment of Duplex PCR for Detecting Aeromonas veronii

作　　者：王惠[1] 边宇[1] 孟庆峰[2] 杨滨僮 康元环[1] 荆琦[3] 单晓枫[1] 钱爱东[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]吉林出入境检验检疫局,吉林长春130062 [3]吉林石油集团有限责任公司洮河农场,吉林松原131100

出　　处：《动物医学进展》2013年第11期16-19,共4页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31201927);国家质检总局科技计划项目(2013IK163)

摘　　要：为了建立维氏气单胞菌双基因PCR检测方法,以维氏气单胞菌ATCC35624基因组DNA为模板,选取16SrRNA和核酸酶基因为靶基因,设计2对特异性引物,建立维氏气单胞菌双基因PCR检测方法。验证方法的敏感性与特异性,同时应用该方法对模拟污染样本进行检测。检测结果显示应用PCR方法同时扩增出大小约880bp和320bp的DNA片段,通过序列比对分析,16SrRNA基因片段、exu基因片段序列与GenBank中登录的维氏气单胞菌ATCC35624株的的同源性均为99%,方法的敏感性较高,能检测到的DNA浓度达到了1.58×10-4 ng/μL,特异性较强,只有维氏气单胞菌标准株及分离株结果呈阳性;人工模拟污染试验显示,该方法的样本检出率达到了90%,高于细菌分离培养的检出率73.3%。建立的维氏气单胞菌双基因PCR检测方法可以克服传统生化鉴定的不足,为维氏气单胞菌的检测提供新的途径。The duplex PCR was developed with genome DNA of Aeromonas veronii ATCC35624 as template and the primers designed according to 16 S rRNA and nuclease gene（exu gene） .The sensitivity and speci-ficity were verified and meanwhile the simulation polluted samples were detected by this method .The re-sults showed that the DNA fragments of approximate 880 bp and 320 bp were obtained .Both 16 S rRNA gene and exu gene shared 99% homology with these genes from Aeromonas veronii ATCC35624 .This method had high sensitivity with the detected DNA concentration approximate 1 .58 × 10-4 ng/μL and high specificity with only the standard strains been detected . The artifical simulation polluted experiments showed that the detected rate of this method with 90% was higher than it of bacterial cultivation with 73 .3% .The developed duplex PCR can overcome the shortage of traditional bacterial cultivation and pro-vide the new method to detect A eromonas veroni .

关 键 词：维氏气单胞菌 16SrRNA 核酸酶基因 聚合酶链反应 

分 类 号：S858.23[农业科学—临床兽医学]