为保障转基因大豆的可追溯性以及消费者的知情权和选择权,开发快速、灵敏、准确的转基因大豆检测方法显得尤为重要。文章建立了一种用于检测转基因大豆及其制品的可视化跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA...为保障转基因大豆的可追溯性以及消费者的知情权和选择权,开发快速、灵敏、准确的转基因大豆检测方法显得尤为重要。文章建立了一种用于检测转基因大豆及其制品的可视化跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)技术。根据抗除草剂草甘膦CP4-EPSPS基因设计引物,进行特异性验证,并对该方法的灵敏度和检出限进行研究。通过检测68份实际样品,对该方法的相对敏感度、相对特异性和相对符合率进行评价。结果表明:可视化SRCA方法具有良好的特异性,其检测转基因大豆的灵敏度为8.8×10^(0)fg/μL,是SRCA凝胶电泳法的10倍,是PCR方法的1000倍。在人工加标样品中,可视化SRCA方法的检出限为0.01%(w/w),均显著低于SRCA凝胶电泳法和PCR方法。与行业检测标准(SN/T 1204—2016)相比,可视化SRCA方法的相对敏感性、相对特异性和相对符合率分别为100.00%、98.41%、98.52%。可视化SRCA方法具有操作简便、灵敏度高、特异性强、检出限低等优点,可以灵敏、高效地检测转基因成分,有利于在检测机构的推广应用。展开更多
The RR soybean was quantitatively detected by ABI Prism 7300 sequence detector with PCR primers and fluorescence probes were designed according to the sequences of endogenous Lectin gene and exogenous CP4-EPSPS gene, ...The RR soybean was quantitatively detected by ABI Prism 7300 sequence detector with PCR primers and fluorescence probes were designed according to the sequences of endogenous Lectin gene and exogenous CP4-EPSPS gene, and the PCR systems were based on SYBR Green I and TaqMan. The standard curve of ACt between CP4-EPSPS gene and Lectin gene of the RR soybean in standard materials was generated and a linear regression equation was obtained. Quantification methods were optimized through two different real-time PCR chemistries, i.e. SYBR Green I and TaqMan, and the RR soybean contents were quantified in five standard samples and seven highly processed products by the two assays. Both methods are proved to be specific, highly sensitive and reliable for both identification and quantification of soybean DNA. The results indicate that the two optimized PCR system can be used for the practical quantitative detection of RR soybean in highly processed products.展开更多
文摘为保障转基因大豆的可追溯性以及消费者的知情权和选择权,开发快速、灵敏、准确的转基因大豆检测方法显得尤为重要。文章建立了一种用于检测转基因大豆及其制品的可视化跨越式滚环等温扩增(Saltatory rolling circle amplification,SRCA)技术。根据抗除草剂草甘膦CP4-EPSPS基因设计引物,进行特异性验证,并对该方法的灵敏度和检出限进行研究。通过检测68份实际样品,对该方法的相对敏感度、相对特异性和相对符合率进行评价。结果表明:可视化SRCA方法具有良好的特异性,其检测转基因大豆的灵敏度为8.8×10^(0)fg/μL,是SRCA凝胶电泳法的10倍,是PCR方法的1000倍。在人工加标样品中,可视化SRCA方法的检出限为0.01%(w/w),均显著低于SRCA凝胶电泳法和PCR方法。与行业检测标准(SN/T 1204—2016)相比,可视化SRCA方法的相对敏感性、相对特异性和相对符合率分别为100.00%、98.41%、98.52%。可视化SRCA方法具有操作简便、灵敏度高、特异性强、检出限低等优点,可以灵敏、高效地检测转基因成分,有利于在检测机构的推广应用。
基金Supported by the Innovative Team Funds of Northeast Agricultural University (CXT004-3-2)Foundation of Heilongjiang Educational Committee(11511030)
文摘The RR soybean was quantitatively detected by ABI Prism 7300 sequence detector with PCR primers and fluorescence probes were designed according to the sequences of endogenous Lectin gene and exogenous CP4-EPSPS gene, and the PCR systems were based on SYBR Green I and TaqMan. The standard curve of ACt between CP4-EPSPS gene and Lectin gene of the RR soybean in standard materials was generated and a linear regression equation was obtained. Quantification methods were optimized through two different real-time PCR chemistries, i.e. SYBR Green I and TaqMan, and the RR soybean contents were quantified in five standard samples and seven highly processed products by the two assays. Both methods are proved to be specific, highly sensitive and reliable for both identification and quantification of soybean DNA. The results indicate that the two optimized PCR system can be used for the practical quantitative detection of RR soybean in highly processed products.
