抗病转基因水稻M12及其产品成分的定性、定量PCR检测方法

通过定性PCR扩增了转基因水稻M12转化体特异性片段和水稻内标基因(sps)片段, 将PCR产物酶切连接后构建到T载体, 得到适于转基因水稻M12品系特异性PCR检测的质粒分子pM12, 建立了事件特异性定性、定量PCR检测方法。定性PCR结果表明, 本方法可特异性检测出M12, 检测限可达100拷贝单倍体水稻基因组; 定量PCR结果表明, M12和sps定量PCR标准曲线R ²为0.998和0.997, 扩增效率为95.3%和108.4%, 重复性分析标准偏差范围为0.043~0.276, 定量极限和检测极限可达100和10拷贝。对转基因含量为1.0%的混合样品定量结果的相对偏差在8.0%以内。本研究建立的方法可用于转基因水稻M12及其加工产品成分的检测。

A Qualitative and Quantitative PCR Detection Method for Disease-resistant Genetically Modified Rice M12 and Its Derivates

In this study, the specific sequence of genetically modified Rice M12 and the endogenous reference gene sps were amplified to construct a T vector as the plasmid pM12 for establishing the qualitative and quantitative PCR detection method of transgenic Rice M12 and its derivates. The qualitative PCR method could specifically quantify the samples of M12 with the detection sensitivity about 100 copies of the rice haploid genome. On the basis of SYBR Green qPCR assay, R ² values of standard curves of M12 and sps were 0.998 and 0.997, the amplification efficiency was 95.3% and 108.4%, respectively. Moreover, the standard deviations (SD) of repeatability ranged from 0.043 to 0.276. The limit of quantification (LOQ) and limit of detection (LOD) were 100 and 10 copies, respectively. The mixed rice sample containing 1.0% gene transforming into rice was exactly quantified by the developed quantitative PCR method, and the quantified bias between the true value and tested value was below 8.0%. In conclusion, these methods can be used for identifying and quantifying M12 and its derivatives.