LIU Wei,YANG Fang,LI Li-hua,et al.Detection of Transgenic Components in Transgenic Soybean by Droplet Digital PCR[J].Soybean Science,2022,41(04):448-454.[doi:10.11861/j.issn.1000-9841.2022.04.0448]
大豆中转基因成分微滴式数字PCR检测方法研究
- Title:
- Detection of Transgenic Components in Transgenic Soybean by Droplet Digital PCR
- 文献标志码:
- A
- 摘要:
- 为了探讨转基因大豆品系及其衍生产品鉴定的高效技术手段，本研究建立了转基因大豆的微滴式数字PCR（ddPCR）筛选检测方法。利用3种方法提取大豆基因组DNA，分析提取方法和保存时间对微ddPCR检测拷贝数的影响。同时，应用特异性引物，以4种转基因大豆筛选靶标元件为扩增靶标，对微滴式数字PCR检测方法进行体系优化、灵敏度测试和准确度验证。结果表明：使用Promega植物基因组DNA提取试剂盒，提取的DNA对转基因作物的外源拷贝数进行鉴定的结果更为准确可靠。与荧光定量筛选方法相比，ddPCR方法更加灵敏和准确。检测体系的最适探针浓度为100 nmol?L-1，最佳退火温度为58 ℃。以4种靶标元件CaMV35S启动子、T-NOS终止子、pat基因和T-E9终止子开展ddPCR检测，检测灵敏度较高，准确性较好。转基因大豆含量为1%时对CaMV35S和T-NOS靶标参数的绝对检测限为2个拷贝，pat为5个拷贝，T-E9为10个拷贝。本研究建立的ddPCR检测方法可用于大豆转化体的定量检测。
- Abstract:
- In order to explore the efficient technical means of identification in transgenic soybean and their derivatives， a droplet digital PCR(ddPCR) screening method was established in this study.We used soybean genomic DNA extracted by three methods to study the effects of extraction quality and preservation time on the copy number identification results of ddPCR.And we carried out the system optimization， sensitivity test and accuracy verification of the ddPCR method by applying four target elements of transgenic soybean as amplification targets. The results showed that it is more accurate and reliable to identify the foreign copy number of transgenic crops by using the DNA extracted from Promega plant genome DNA extraction kit. Compared with fluorescence quantitative screening， ddPCR was more sensitive and accurate. The optimum concentration of probe was 100 nmol?L-1 and the optimum annealing temperature was 58 ℃. Four target elements， CaMV35S promoter， T-NOS terminator， pat gene and T-E9 terminator， were detected by ddPCR with high sensitivity and accuracy. The absolute limit of detection(LOD) for CaMV35S and T-NOS target parameters was two copies， five copies for pat and ten copies for T-E9 when the content of transgenic soybean was 1%. The ddPCR method established in this study can be used for quantitative detection of soybean transformants.
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备注/Memo
收稿日期：2021-11-15