数字PCR法定量低浓度水平短链DNA标准物质的研究

Quantification of short length and low concentration level DNA reference material based on digital PCR

该文研制一种低浓度水平的短链DNA标准物质,用于低浓度水平ctDNA检测过程的量值溯源,重点对低浓度水平的短链基因标准物质的定值方法进行研究。首先选取TP53基因的一段300 bp长度的序列作为标准物质的目标序列,以数字PCR方法为研究方法,优化引物探针设计,通过考察PCR扩增的效率可以达到98.6%,满足数字PCR方法扩增的要求;然后采用转基因玉米NK603质粒分子国家标准物质验证数字PCR方法定值的准确度和精密度;其次考察数字PCR方法定量低浓度短链DNA标准物质TP53的重复性和重现性分别为2.99%和5.23%;最后采用数字PCR方法8家单位联合定值确定标准物质TP53的终浓度为(613.85±9.12)copies/μL(k=2),实现了对低浓度水平短链DNA标准物质的定值。

In this work,a short-length and low concentration level DNA reference material is developed,which is used to guarantee the reliability and traceability of low-concentration level ctDNA detection process,focusing on the quantification method of short-length and low concentration level DNA reference material.Firstly,A 300 bp fragment of TP53 gene was selected as target,a digital PCR(dPCR)method was used as a research method,primers and probes of the target were designed and optimized.The efficiency of PCR amplification was investigated and the result arrived to 98.6%,satisfying the requirement of the amplification of dPCR.Secondly,the accuracy and precision of the digital PCR method were verified by national standard reference transgenic corn NK603 plasmid molecular.Thirdly,the repeatability and reproducibility of the shortlength and low concentration level DNA reference material TP53 were 2.99%and 5.23%,respectively.Finally,the concentration of the TP53 was certified using digital PCR method in 8 different laboratories,and the uncertainty evaluation was performed.The final concentration of the TP53 was(613.85±9.12)copies/μL(k=2).