旋毛虫Ts43基因和内参基因标准品质粒及标准曲线的构建

Construction of standard plasmid DNA and standard curve for Ts43 gene and reference gene in Trichinella spiralis

为了构建旋毛虫Ts43基因和内参基因18S rRNA基因标准品和标准曲线,试验采用RT-PCR方法对目的基因进行扩增,产物纯化后与pGEM-T-easy载体连接,转化到宿主菌DH5α中,筛选后得到重组质粒,对重组质粒进行酶切、质粒PCR和测序鉴定,经鉴定后的重组质粒作为标准品进行荧光定量PCR,以制定标准曲线。结果表明:成功克隆了旋毛虫Ts43和18S rRNA基因片段,应用重组质粒制定的定量曲线循环阈值与模板浓度具有良好的线形关系,回归系数分别为0.999和0.992。说明试验成功构建了Ts43和18S rRNA标准品质粒和标准曲线。

To construct standard plasmid DNA and standard curve of real-time PCR for Ts43 gene and reference gene in Trichinella Spiralis,total RNA was extracted from muscle larvae in Trichinella Spiralis and the fragments of target gene were amplified by RT-PCR.The products were ligated into a pGEM-T Easy vector and the ligated vectors were transformed into competent cells of Escherichia coli DH5α.The clones were identified by digesting the plasmid DNA with enzyme,PCR and sequencing.The results showed that the positive plasmids were used to construct a calibration curve,the relative standard curve method was shown to be of high linearity and the linearity is 0.999 and 0.992,respectively.The standard plasmid DNA and standard curve for Ts43 gene and 18S rRNA gene were successfully constructed.