实时荧光PCR检测蜡样芽胞杆菌重组质粒标准的构建

Construction of Recombinant Plasmid as the Standards for the Detection of Bacillus cereus with Real-time Quantitative Polymerase Chain Reaction

〔目的〕构建蜡样芽胞杆菌16s-23sITS重组质粒,为实时荧光PCR检测蜡样芽胞杆菌提供质粒标准。〔方法〕选择蜡样芽胞杆菌16s-23sITS特异基因作为目的靶序列,设计、合成引物和探针,采用普通PCR扩增特异靶序列,克隆到pGM-T载体后,转化感受态大肠埃希菌DH5,α经蓝白斑筛选,再分别用EcoRI酶切,普通PCR及测序3种方法证实目的片段已成功重组。建立荧光定量PCR检测蜡样芽胞杆菌的标准曲线。〔结果〕成功构建目的重组质粒,并以此为标准制作荧光定量PCR标准曲线,重组质粒标准具有较大的线性范围(102copies/μl～108copies/lμ)和灵敏度。〔结论〕该方法构建的16s-23sITS基因重组质粒能满足实时荧光定量测定对参考标准的要求,为后续实时荧光PCR快速检测蜡样芽胞杆菌的推广应用提供了条件。

Objective To construct the 16s-23s ITS recombinant plasmid as the DNA standards for the detection of Bacillus cereus with real-time quantitative polymerase chain reaction （q-PCR）. Methods 16s-23s ITS gene was selected as the detection target and the primers and the probe were designed correspondingly. After being amplified by PCR, the target gene was ligated with pGM-T vector. Then the product of ligation was transformed into the E.Coli DH5a competent cells. Plasmids were extracted from positive clones and confirmed with PCR, EcoR I digestion and sequencing. At last a standard curve was established for the rapid detection of Bacillus cereus with q-PCR. Results The standard curve for rapid detection of Bacillus cereus via 16s-23s ITS gene was successfully established with the wide detection range （10^2copies/ul-10^^8 copies/ul） and high sensitivity. Conclusion The recombined plasmid standard can satisfy the need of experiments.And it establishes the foundation for the rapid detection of Bacillus cereus with the real-time PCR.