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为建立快速检测框镜鲤致病性维氏气单胞菌(A.veronii)双重PCR方法,本研究以A.veronii CY0806株和国际标准株DNA为模板,分别以16S rRNA和Aer基因特异性引物进行PCR扩增,分别获得大小约880 bp和430 bp的DNA片段。通过序列比对分析,16S rRNA基因片段、Aer片段序列与GenBank中登录的A.veronii ATCC35624株的的同源性均为99%。进一步试验显示该方法的敏感性较高,达到1.58×10-3ng/μL,特异性较强,只有A.veronii标准株及分离株结果呈阳性;人工模拟污染样本试验显示:该方法的检出率达到了86.7%,高于细菌分离培养的70%检出率。双重PCR检测方法的建立,为框镜鲤致病性A.veronii的检测提供新的方法。
In order to establish the double-PCR method for the rapid detection of A.veronii in the test carp, A.veronii CY0806 strain and the international standard strain DNA were used as template to analyze the 16S rRNA and Aer gene-specific The primers were used for PCR amplification to obtain DNA fragments of about 880 bp and 430 bp respectively. By sequence alignment analysis, 16S rRNA gene fragment and Aer fragment sequence shared 99% identity with A.veronii ATCC35624 strain registered in GenBank. Further tests showed that the method was highly sensitive, reaching 1.58 × 10-3ng / μL, specificity is strong, only A.veronii standard strains and isolates positive results; artificial simulated contamination of the sample test showed that: the method was detected The rate reached 86.7%, higher than 70% of bacteria isolated culture detection rate. The establishment of double PCR detection method provides a new method for the detection of pathogenic A.veronii in the mirror carp.