中国农业科学 ›› 2021, Vol. 54 ›› Issue (22): 4728-4739.doi: 10.3864/j.issn.0578-1752.2021.22.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

转基因玉米NK603转化体/zSSIIb内标基因二重微滴数字PCR方法的建立及应用

肖芳1(),李俊1,王颢潜2,翟杉杉1,陈子言2,高鸿飞1,李允静1,吴刚1,张秀杰2(),武玉花1()   

  1. 1中国农业科学院油料作物研究所/农业农村部油料作物生物学与遗传育种重点实验室,武汉 430062
    2农业农村部科技发展中心,北京 100025
  • 收稿日期:2021-04-28 接受日期:2021-06-16 出版日期:2021-11-16 发布日期:2021-11-19
  • 通讯作者: 张秀杰,武玉花
  • 作者简介:肖芳,E-mail: xf_hzau@163.com
  • 基金资助:
    转基因生物新品种培育专项(2016ZX08012003)

Establishment and Application of A Duplex ddPCR Method to Quantify the NK603/zSSIIb Copy Number Ratio in Transgenic Maize NK603

XIAO Fang1(),LI Jun1,WANG HaoQian2,ZHAI ShanShan1,CHEN ZiYan2,GAO HongFei1,LI YunJing1,WU Gang1,ZHANG XiuJie2(),WU YuHua1()   

  1. 1Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement Oil Crops Ministry of Agriculture and Rural Affairs, Wuhan 430062
    2Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs of People’s Republic of China, Beijing 100025
  • Received:2021-04-28 Accepted:2021-06-16 Online:2021-11-16 Published:2021-11-19
  • Contact: XiuJie ZHANG,YuHua WU

摘要:

【目的】批准进口的转基因玉米NK603是中国转基因生物安全监管的主要对象。转基因安全监管需要标准物质和标准检测方法,建立转基因玉米NK603的数字PCR(dPCR)方法将为转基因玉米NK603的定量检测和标准物质研制提供精准测量技术。【方法】采用人工合成技术构建标准质粒分子pUC57-NK603;将NK603转化体与不同的玉米内标基因引物/探针一一组合,遴选与NK603转化体特异性PCR具有相同扩增能力的玉米内标基因PCR方法;设置二重微滴数字PCR(ddPCR)的退火温度梯度和引物/探针浓度梯度,优化二重ddPCR的反应体系和反应条件;用梯度稀释的标准质粒溶液作模板,考察二重ddPCR的检测极限、定量极限和动力学范围;将转基因玉米NK603种子粉末和非转基因玉米种子粉末混合,配制质量分数分别为100%、10%和6%的盲样,考察二重ddPCR的定量准确性。【结果】用标准质粒分子pUC57-NK603作为二重ddPCR的质控对照,通过考察二重ddPCR反应热图的微滴信号强度、阳性微滴与阴性微滴分辨率、雨滴数量、NK603转化体与内标基因拷贝数比值测量值与预期值的一致性,确定将NK603转化体特异性PCR方法与内标基因zSSIIb PCR方法组合,建立NK603/zSSIIb二重ddPCR方法。二重ddPCR反应体系中NK603转化体和zSSIIb内标基因的引物/探针浓度相同,均为400 nmol·L-1/200 nmol·L-1,在60℃退火延伸。NK603/zSSIIb二重ddPCR的检测极限是2 copies DNA模板,定量极限是48 copies DNA模板,动力学范围是10—60 000 copies DNA。应用NK603/zSSIIb二重ddPCR方法可准确定量玉米盲样中的NK603转化体含量,定值结果变异系数小于25%;dPCR定量结果与荧光定量PCR(qPCR)定量结果无显著差异,且具有更高的精确性。【结论】内标基因的选择会影响dPCR定量结果的准确性,在建立dPCR方法的过程中,要用具有准确量值的样品作为质控对照,评估内标准基因的适用性。以人工合成的标准质粒分子pUC57-NK603为质控对照,建立了NK603/zSSIIb二重ddPCR方法。应用建立的二重ddPCR定值方法进行标准物质的研制和定值,已成功研制出转基因玉米NK603有证标准物质。

关键词: 转基因玉米NK603, 二重数字PCR, 标准质粒分子, 内标基因, 定量

Abstract:

【Objective】Transgenic maize NK603 approved for import is an important target of genetically modified organism (GMO) regulation in China. The implementation of GMO regulation requires reference materials (RMs) and standardized detection methods. Establishment of a duplex droplet digital PCR (ddPCR) would provide accurate measurement technology for quantification of NK603 event and development of NK603 RMs. 【Method】 A standard plasmid molecule pUC57-NK603 was constructed by DNA synthetic technique; the primer/probe set of NK603 event was combined with different maize reference genes one by one to select reference gene PCR assay with identical amplification ability to NK603 event-specific PCR assay; main reaction parameters, such as annealing temperature and primer/probe concentration,were optimized in the course of establishing duplex ddPCR; the standard plasmid solution was serially diluted to investigate the limit of detection (LOD), limit of quantification (LOQ) and dynamic range of the duplex ddPCR assay; blinded samples with mass fractions of 100%, 10% and 6% were prepared by mixing NK603 powder with non-GM counterpart, to evaluate the accuracy of quantitative results of the duplex ddPCR. 【Result】 The reference gene zSSIIb was determined to combine with NK603 event to establish the NK603/zSSIIb duplex ddPCR method with the standard plasmid molecule pUC57-NK603 as a quality control after analyzing the fluorescence amplitude of positive droplets, separation between positive and negative droplets, raindrop number, and consistency between measured copy number ratio and expected copy number ratio of NK603 event to reference gene. The primer/probe concentration was optimized to be 400 nmol·L-1/200 nmol·L-1 for both NK603 event and zSSIIb gene, and the annealing temperature was determined to be 60°C. The LOD of NK603/zSSIIb duplex ddPCR was estimated to be 2 copies of DNA template, the LOQ was 48 copies of DNA template, both NK603 assay and zSSIIb assay showed good linearity between measured values and theoretical values over the dynamic range from 10 to 60 000 copies of DNA template. The NK603/zSSIIb duplex ddPCR achieved accurate quantitative results of NK603 content in blind maize samples with less than 25% of coefficient of variation; the quantitative results of ddPCR were not significantly different from those of real-time quantitative PCR (qPCR), moreover, the duplex ddPCR showed an advantage over qPCR in term of precision. 【Conclusion】 The selection of reference genes affects the accuracy of quantitative results by ddPCR. Establishment of ddPCR methods should use samples with accurate GMO content as quality controls to evaluate the applicability of reference genes. The NK603/zSSIIb duplex ddPCR method was successfully established using the synthetic standard plasmid molecule pUC57-NK603 as a quality control. NK603 certified reference materials (CRMs) have been successfully developed in China by applying the established NK603/zSSIIb duplex ddPCR assay to characterize the property values.

Key words: transgenic maize NK603, duplex droplet digital PCR, reference plasmid molecule, reference gene, quantification