轮状病毒荧光定量PCR标准品的构建

摘要

To construct standard for detection of rotavirus in environmental water using the quantitative real⁃time polymerase chain reaction(PCR),rotavirus standard for the real⁃time assay is prepared by MA104 cell culturation,PCR and T⁃A clone methods with primers specific for the viral structure protein of VP4 gene,and this standard is confirmed by enzyme cleavage and DNA sequencing. Specificity, stability and reproducibility of the standard quantified are detected by common and real⁃time fluorescence⁃labeled quantitative PCR. The result of standard curve shows an excellent linear negative regression(the slope is-4.015;R2=0.991)between the threshold cycle Cvand Log starting quantity of copy number. The melting curve analysis of real⁃time PCR shows melting temperature at 85.8℃,indicating that PCR products are the rotavirus VP4 sequence and thus the standard used in this research is specific for rotavirus. Moreover,the result of real⁃time PCR also indicates detection range is 5×101-5×1010 copies/μL,and the detection limit for this assay is 50 copies of rotavirus per copy concentration gradient reaction,and thus real⁃time PCR assay using the standard has a high sensitivity for detection of rotavirus.Furthermore,the result indicates a high reproducibility and stability of plasmid standard according to Cv(Cv<1.5%)of three independent experiments. Taken together,in this research,rotavirus plasmid standard prepared could be used as quantitative detection of rotavirus from water samples.%　　采用MA104细胞系培养和T载体克隆技术，在轮状病毒结构蛋白VP4基因序列中设计合成引物，经过PCR扩增后将特异性产物与pMD-19-T载体连接，对获得的轮状病毒重组质粒标准品进行酶切鉴定和测序分析。利用常规PCR和荧光定量PCR方法对获得的重组质粒标准品进行特异性和灵敏度指标的检验。结果表明，将构建的重组质粒作为标准品制备标准曲线具有较高的扩增效率和良好的线性关系（斜率为-4.015， R2=0.991）；荧光定量PCR熔解曲线分析表明，85.8℃的PCR产物是轮状病毒VP4基因序列的特异性产物，该标准品具有轮状病毒特异性；同时，所制备的重组质粒标准品在荧光定量PCR检测中具有较大的线性范围（5×101~5×1010拷贝/μL），每个梯度PCR反应最低可以检测到50拷贝的重组质粒，因而采用该标准品进行荧光定量PCR分析具有较高的检测灵敏度；此外，该重组质粒标准品具有较高的稳定性和重复性，3次独立实验的变异因数Cv<1.5%。构建的重组质粒标准品可以用做环境水体中轮状病毒的荧光定量PCR标准品。