牛种布鲁氏菌A19-△VirB12标记疫苗双重实时荧光定量PCR方法的建立

Establishment of a dual real-time fluorescent PCR method for detecting the Brucella abortus A19-△VirB12 mutant vaccine strain

目的建立一种区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒感染株的双重荧光定量PCR方法。方法分别以布鲁氏菌4型分泌系统中VirB8基因、VirB12基因序列设计2对引物及探针,优化实时荧光PCR反应体系及条件。以牛种布鲁氏菌A19-△VirB12标记疫苗株、牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株、大肠杆菌、沙门氏菌基因组DNA进行Realtime-PCR扩增,评价该方法特异性。分别构建布鲁氏菌VirB12基因和VirB8基因片段阳性质粒,10倍系列稀释后进行Realtime-PCR扩增,测定该方法的敏感性。结果本方法具有良好的特异性,牛种布鲁氏菌A19疫苗株、羊种布鲁氏菌M5疫苗株以及猪种布鲁氏菌S2疫苗株基因组DNA同时出现VirB8基因与VirB12基因阳性扩增,牛种布鲁氏菌A19-△VirB12标记疫苗株仅出现VirB8基因阳性扩增,大肠杆菌、沙门氏菌均未扩增出目的条带,对VirB8基因及VirB12基因片段阳性质粒的检测限分别为约10^(2) copies/μL和10^(3) copies/μL。该方法仅用于鉴别区分牛种布鲁氏菌A19-△VirB12标记疫苗株与布鲁氏菌野毒株。结论本研究建立的布鲁氏菌双重Realtime-PCR方法,具有良好的特异性和敏感性,为今后鉴别牛种布鲁氏菌A19-△VirB12分子标记疫苗免疫牛与自然感染牛提供技术支撑。

This study aimed to establish a dual-fluorescence quantitative PCR method to differentiate between the Brucella abortus A19-△VirB12 mutant vaccine strain and the wild virus infection strain.Two pairs of specific primers and two probes were designed on the basis of VirB8 from the type IV secretion system of Brucella and the VirB12 gene deletion sequence of the Brucella abortus A19-△VirB12 vaccine strain.After screening of the primers and optimization of both the reaction system and conditions,the genomic DNA of Brucella abortus A19-△VirB12 mutant vaccine strain,Brucella abortus A19 vaccine strain,Brucella melitensis M5 vaccine strain,Brucella suis S2 vaccine strain,Escherichia coli and Salmonella were amplified by real-time PCR to evaluate the specificity of the method.Plasmids positive for the VirB12 gene and VirB8 gene fragments of Brucella were constructed.After 10 fold serial dilution,real-time PCR amplification was performed to determine the sensitivity of the method.The results indicated high specificity.The VirB8 gene and VirB12 gene were simultaneously amplified in the genomic DNA of the Brucella abortus A19 vaccine strain,Brucella melitensis M5 vaccine strain and Brucella suis S2 vaccine strain.The VirB8 gene was amplified for only the Brucella abortus A19-△B12 mutant vaccine strain,whereas no amplification of Escherichia coli and Salmonella was observed.The detection limits of this method for the VirB8 gene and VirB12 gene were 10^(2) copies/μL and 10^(3) copies/μL respectively.In summary,a dual-fluorescence quantitative PCR method with high sensitivity and specificity was established,thus supporting the future identification of Brucella A19-△B12 mutant vaccine immunized cattle and wild virus infected cattle.