CAJ | 학술논문

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16 S rRNA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880 bp和320 bp的DNA片段，通过序列比对分析，16 S rRNA基因片段、exu基因片段序列与GenBank中登录的维氏气单胞菌A T CC35624株的的同源性均为99％，方法的敏感性较高，能检测到的DN A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。
위료건립유씨기단포균쌍기인PCR검측방법，이유씨기단포균ATCC35624기인조DNA위모판，선취16 S rRNA화핵산매기인위파기인，설계2대특이성인물，건립유씨기단포균쌍기인PCR검측방법。험증방법적민감성여특이성，동시응용해방법대모의오염양본진행검측。검측결과현시응용PCR방법동시확증출대소약880 bp화320 bp적DNA편단，통과서렬비대분석，16 S rRNA기인편단、exu기인편단서렬여GenBank중등록적유씨기단포균A T CC35624주적적동원성균위99％，방법적민감성교고，능검측도적DN A농도체도료1．58×10-4 ng/μL ，특이성교강，지유유씨기단포균표준주급분리주결과정양성；인공모의오염시험현시，해방법적양본검출솔체도료90％，고우세균분리배양적검출솔73．3％。건립적유씨기단포균쌍기인PCR검측방법가이극복전통생화감정적불족，위유씨기단포균적검측제공신적도경。
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 .