数字PCR方法准确测量质粒DNA拷贝浓度

Accurate Plasmid DNA Copy Concentration Quantification by Digital PCR

中国计量科学研究院(NIM)利用数字PCR技术针对国际比对样品质粒DNA,通过设计2对特异性的引物和探针、优化PCR扩增体系、排除PCR mastermix中的DNA污染,成功建立了定量该质粒DNA的数字PCR方法。比较了采用单重PCR和双重PCR方法定量结果之间的差异,最终实现了对比对样品的定量和不确定度评定。对线性质粒样品的测量结果及其扩展不确定度(k=2)为(8.06±0.55)×10~3 copies/nag,该结果在比对参考值不确定度范围内,且与参考值非常接近。

NIM established two digital polymerase chain reaction （dPCR） assays for quantifying the plasmid DNA sample, including designing two pairs of specific primer and probe, optimizing PCR amplifying conditions and excluding the DNA contamination in the PCR mastermix. The measurement results by simplex and duplex dPCR are compared. The DNA sample is successfully quantified by the established dPCR method and the measurement uncertainty is eventually evaluated. The measurement result with its expanded uncertainty （k = 2 ） is （8.06±0.55）×10^3 copies/mg, it agree well with the reference value within the standard uncertainty and very close to the reference value.