反复冻融质粒标准品对荧光实时定量PCR实验的影响

Impact of repeated freezin g and thawing of plasmid standards for real-time fluorescent quantitative PCR

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、1.0×104和1.0×102拷贝/μl高中低3个浓度的标准品,反复冻融1、6、11、15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。结论大于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。

Aim To observe the impact of repeated freezing and thawing of plamid standards on the quality of of real-time PCR detection. Methods The cells of E. coli DH5α clone abundance of the recombinant plasmid pMD -18T,quantitated by UV spectrophotometer,based on calculated molecular weight of the recombinant plasmid copy number,diluted into a concentration gradient of the fluorescence of the standard real-time quantitative PCR products,high （1.0×10^6copies/ μl）,middle（1.0×10^4copies / μl）and low（1.0×10^2copies / μl）concentrations for performing real time PCR amplification and quantitating tests after repeated freeze-thaw once,6 times,11 times,15 times and 21 times of the standard plasmid as a template. Results The value of the stability of high and low concentrations of relatively great fluctuations,the great variation of high plasmid concentration was at the magnitude of 0.06 after freezing and thawing 21 times; the great changes of the plamid concentration was at the magnitude of 0.18; the changes of low plasmid concentration was in magnitude of 0.34. Conclusion The middle an high concentrations of plasmid standards of 1.0×104copies / μl can tolerate repeated freezing and thawing of 21 times,while relatively great changes have been oberved on the plasmid concentration lower than 1.0 × 10c4opies / μl. Thus repeated freezing and thawing of plasmid at low concentration is not recommended..