水中轮状病毒实时定量PCR外标准品的构建

Construction of External Standard for Detection of Rotavirus in Water Using the Quantitative Real-Time Polymerase Chain Reaction

采用细胞培养和T-A克隆技术,在轮状病毒主要结构蛋白VP7基因序列上设计合成引物,经PCR扩增后将特异性产物连接入pGEM-T-easy载体中,经过酶切鉴定和测序分析获得轮状病毒cDNA标准品.利用常规PCR和实时定量PCR方法对所获得的cDNA标准品进行特异性、稳定性和重复性指标的检验.结果表明,利用此标准品制备的标准曲线具有较高的扩增效率和良好的线性关系（斜率为-3.353,R^2=0.995）;实时定量PCR熔解曲线分析表明,温度在81℃±0.5℃的PCR产物是轮状病毒VP7序列上的特异性产物,表明此标准品是轮状病毒特异性的;同时,所制备的标准品在实时定量PCR检测中具有较大的线性范围（9×10^0～9×10^11copies/μL,每个反应最低可以检测到9个拷贝数的轮状病毒cDNA,表明利用该标准品进行实时定量PCR分析时具有较高的检测灵敏性;此外,该标准品具有较高的稳定性和重复性（3次独立实验CV值在0.2%～0.9%之间）,可长期稳定保存.构建的标准品可用作检测水环境中轮状病毒的实时荧光定量PCR的外标准品.

To construct external standard for detection of rotavirus in water using the quantitative real-time polymerase chain reaction, rotavirus eDNA standard for real-time PCR assay was prepared by cell culture, PCR and T-A clone methods with primers specific for the viral structure protein VP7 gene, and this eDNA standard was confirmed by enzyme cleavage and DNA sequencing. Specificity, stability and reproducibility of the cDNA standard quantified were detected by common polymerase chain reaction （PCR） and quantitative real-time PCR. The results of standard curve showed a very good linear negative regression between threshold cycle （Ct） and Log starting quantity of copy number. The melting curve analysis of real-time PCR showed melting temperature at 81 ℃ ± 0.5℃, indicating PCR products were that of the rotavirus VP7 sequence and thus the standard used in this study was specific for rotavirus. Moreover, the result of real-time PCR also indicated detection range was from 9 × 10^ 0to 9 × 10^11 copies per reaction, and the detection limit for this assay was 9 copies of rotavirus eDNA per reaction, and thus real-time PCR assay using the standard had a high sensitivity for detection of rotavirus. Furthermore, the results indicated a high stability and reproducibility of eDNA standard were assessed according to the CVs of three independent experiments in the range of 0.2% ～ 0.9%. Taken together, in this study rotavirus eDNA standard prepared could be used as quantitative detection of rotavirus eDNA from water samples.