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数字PCR法定量低浓度水平短链DNA标准物质的研究

281    2020-01-19

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作者:闻艳丽, 梁文, 刘刚

作者单位:上海市计量测试技术研究院化学与电离辐射计量研究所, 上海 201203


关键词:DNA标准物质;TP53基因;低浓度水平;数字PCR法


摘要:

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。


Quantification of short length and low concentration level DNA reference material based on digital PCR
WEN Yanli, LIANG Wen, LIU Gang
Division of Chemistry and Ionizing Radiation Measurement Technology, Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China
Abstract: 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 short-length 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).
Keywords: DNA reference material;gene TP53;low concentration level;digital PCR
2020, 46(1):44-49  收稿日期: 2019-06-17;收到修改稿日期: 2019-07-29
基金项目: “国家质量基础的共性技术研究与应用”重点专项(2017YFF0204605);国家自然科学基金面上基金(21775104)
作者简介: 闻艳丽(1984-),女,湖北宜昌市人,高级工程师,博士,研究方向为生物计量
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