公共引物介导的多重定量PCR技术在转基因大豆检测中的应用研究

Application of public primer-mediated multiplex quantitative PCR technology in the detection of genetically modified soybeans

中文关键词:  转基因大豆  基因表达平台  多重PCR  公共引物

英文关键词:genetically modified soybean  gene expression platform  multiplex PCR  public primer

中文摘要:

      目的 建立公共引物介导的多重定量PCR法检测我国农业农村部允许进口的6种转基因大豆(包括: MON87701、DP356043、CV-127、MON87769、MON87705、MON87708)的方法。方法 设计并筛选出针对本研究涉及的6种转基因大豆对特异性引物, 在每对特异性引物的5’端均加上一段公共引物, 形成特异性嵌合引物。以某种转基因大豆DNA为模板, 加入多引物体系以及荧光公共引物验证GeXP(gene expression platform)多重检测体系的特异性, 每种转基因大豆DNA模板浓度分别稀释为20、2、1、0.2、0.02 ng/μL, 运用GeXP多重PCR方法进行单一DNA模板灵敏度分析并优化各对特异性嵌合引物的工作浓度。结果 多重PCR检测体系扩增出特异性片段, GeXP检出的各转基因大豆片段长度MON87701、DP356043、CV-127、MON87769、MON87705和MON87708分别为140.75、184.42、274.93、308.47、467.98和517.67 bp, GeXP 多重检测体系检测敏感性可达到为1 ng/μL, 扩增产物测序结果进一步说明了引物扩增的特异性。结论 本研究建立的基于GeXP系统的多重PCR定量检测技术, 可以准确鉴别6种转基因大豆, 为转基因大豆及其他转基因产品提供了新型的检测方法。

英文摘要:

      Objective To establish a method for the determination of 6 kinds of transgenic soybeans (including: MON87701, DP356043, CV-127, MON87769, MON87705 and MON87708) allowed to be imported by the Ministry of Agriculture and Rural Affairs of China by public primer-mediated multiple quantitative PCR. Methods Pair specific primers were designed and screened for 6 kinds of transgenic soybean involved in this study. A public primer was added to the 5’ends of each pair of specific primers to form specific chimeric primers. A transgenic soybean DNA was used as a template, multiple primer systems and fluorescent public primers were added to verify the specificity of the multiple detection system of gene expression platform (GeXP). The concentration of each transgenic soybean DNA template was diluted to 20, 2, 1, 0.2, 0.02 ng/μL, respectively. The sensitivity of the single DNA template was analyzed by GEXP multiplex PCR method and the working concentration of each pair of specific chimeric primers was optimized. Results The specific fragments were amplified by multiple PCR detection system, and the length of the transgene soybean fragments MON87701, DP356043, CV-127, MON87769, MON87705 and MON87708 detected by GeXP was 140.75, 184.42, 274.93, 308.47, 467.98 and 517.67 bp, respectively. The sensitivity of Gexp multiple detection system was up to 1 ng/μL. Sequencing results of the amplified products further demonstrated the specificity of primer amplification. Conclusion The multiple PCR quantitative detection technology based on GEXP system established can accurately identify 6 kinds of transgenic soybeans, which provides a new method for the detection of transgenic soybeans and other transgenic products.