反复冻融对荧光实时定量PCR质粒标准品的影响研究.docVIP

反复冻融对荧光实时定量PCR质粒标准品的影响研究来源：中国论文下载中心??? - ????????????????????? 作者：王亨莉，陈红涛，孔梅，杨冬铭? 【摘要】? 目的 评价反复冻融对不同浓度的real-time PCR质粒标准品的影响。方法 用大肠杆菌DH5α感受态细胞克隆丰量的pMD-18T重组质粒，紫外分光光度计定量后，根据重组质粒分子量计算其拷贝数，稀释成梯度浓度的荧光实时定量 PCR的标准品，取1.0×106拷贝/μl、1.0×104拷贝/μl 和1.0×102拷贝/μl高中低3个浓度的标准品，反复冻融1次，6次，11次，16次和21次后，以冻融后的质粒标准品为模板同时进行real-time PCR扩增定量，比较量值变化。结果 反复冻融21次以内，高中值稳定，低浓度波动相对较大，高浓度质粒最大变化量为0.06个数量级;中浓度质粒最大变化量为0.18个数量级;低浓度质粒最大变化量为0.34个数量级。结论 1.0×104拷贝/μl的高中浓度的质粒标准品可以耐受21次以内反复冻融，低于1.0×104拷贝/μl的质粒标准品反复冻融后量值变化较大，临床使用的试剂盒所配的低浓度标准品应避免反复冻融。 【关键词】? 实时定量 PCR;质粒DNA标准品 　　【Abstract】 Objective To evaluate freezing and thawing repeatedly influencing plasmid DNA standards for fluorescence quantification real-time PCR.Methods The recombinant PMT-18 plasmid was cloned in the Escherichia coli DH5a cells. The concentrations of plasmid stocks were determined by the Eppendorf Biophotometer, and the copy number of each plasmid was calculated based on its molecular weight. Plasmid standards were made by preparing serial 10-fold dilutions, the plasmid standards were stored at -20℃. After freezing and thawing plasmid standards 1, 6, 11, 16 and 21 times repeatedly, quantity value diversity of different concentration standards was confirmed by real-time PCR.Results Freezing and thawing less than 21 times, and there was no significant change observed in high and mid concentrations of plasmid standards, but low concentrations of plasmid standards was varied. The largest variation of high concentration plasmids was 0.06 lg, the largest variation of mid concentration plasmids was 0.18 lg, and the largest variation of low concentration plasmids was 0.34 lg.Conclusion Plasmid standards more than 1.0×104copies/μl is stable within freezing and thawing 21 times, plasmid standards less than 1.0×104copies/μl is affected after freezing and thawing repeatedly, the low concentration standard in clinical diagnosis kit should avoid freezing and thawing repeatedly. 　　【Key words】 real-time quantitative PCR; plasmid DNA standard 　　基于Taq