HSA转基因水稻二重微滴数字PCR定量检测方法研究  

Study on double microdroplet digital PCR for accurate quantitative detection of the transgenic rice expressing human serum albumin

作　　者：李晓飞[1] 姚晓青 闫晓红[1] 李俊[1] 朱莉[1] 吴刚[1] 罗军玲[1] LI Xiao-fei;YAO Xiao-qing;YAN Xiao-hong;LI Jun;ZHU Li;WU Gang;LUO Jun-ling(Key Laboratory of Biology and Genetic Improvement of Oil Crops,Ministry of Agriculture and Rural Affairs/Oil Crops Research Institute,Chinese Academy of Agricultural Sciences,Wuhan 430062,China)

机构地区：[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,湖北武汉430062

出　　处：《中国油料作物学报》2022年第5期1098-1107,共10页Chinese Journal of Oil Crop Sciences

基　　金：转基因生物新品种培育专项(2016ZX08012-003)。

摘　　要：重组人血清白蛋白(HSA,Human serum albumin)基因工程水稻是我国自主研发的可规模化生产水稻重组人血清白蛋白(OsrHSA)的转基因品系,具有极高的推广价值和应用前景,但是目前缺乏对其进行精准定量检测的方法。微滴数字PCR(ddPCR,droplet digital PCR)是近年来新兴的前沿PCR技术,不依赖标准物质即可实现对DNA分子的绝对定量,在转基因产品定量领域被广泛应用。本研究基于ddPCR平台建立了适用于重组人血清白蛋白基因工程水稻(114-7-2品系)的二重ddPCR精准定量检测方法。通过对引物探针的特异性进行验证、对引物探针工作浓度和反应退火温度进行优化,获得最佳反应条件。进而对该方法检测限、定量限和结果重复性做了测定。最后通过对不同含量的重组人血清白蛋白转基因水稻样品进行定量检测,分析比较传统实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和二重ddPCR的优劣,结果表明本研究建立的转基因水稻HSA/PLD二重ddPCR方法稳定性更好、灵敏度高、成本低廉,精准可靠,适用于不依赖标准物质的精准定量HSA转基因水稻转化体含量分析,可取代qRT-PCR方法进行HSA转基因水稻的绝对定量检测,完善了我国转基因水稻成分精确定量检测技术体系。Gene engineering rice expressing Oryza sativa recombinant Human Serum Albumin(OsrHSA),is a transgenic rice line independently developed in China that can produce OsrHSA in rice on a large scale.It has high promotion value and application prospect,but there is no accurate and quantitative detection method for it at present.Droplet digital PCR(ddPCR)is an emerging frontier PCR technology in recent years,which can achieve the absolute quantification of DNA molecules without relying on standard substances,and has been widely used in the field of quantification of transgenic products.In this study,a dual ddPCR method for accurate quantitative detection of recombinant human serum albumin gene engineering rice(Line 114-7-2)was established based on ddPCR platform.The optimal reaction conditions were obtained by verifying the specificity of primer probe,optimizing the concentration of primer probe and annealing temperature.Furthermore,the limits of detection,quantification and reproducibility of the method were determined.Finally,through quantitative detection of rice samples of different contents of transgenic rice within,the advantages and disadvantages of quantitative real-time PCR(qRT-PCR)and dual ddPCR were analyzed and compared.The results showed that the HSA/PLD dual ddPCR method established in this study had better stability,high sensitivity,low cost,accuracy and reliability,and was suitable for accurate quantitative analysis of transgenic rice expressing OsrHSA independent of standard substances.It could replace qRTPCR method for absolute quantitative detection of transgenic rice expressing OsrHSA.The technical system for accurate and quantitative determination of transgenic rice components in China was improved.

关 键 词：基因工程水稻 水稻重组人血清白蛋白(OsrHSA) 微滴数字PCR 绝对定量 转基因 

分 类 号：Q78[生物学—分子生物学] S511[农业科学—作物学]