抗病转基因水稻M12及其产品成分的定性、定量PCR检测方法  

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

作　　者：李鹏[1,2] 张琳 叶吉妮 贺诗瑶 贾军伟[1] 潘爱虎[1] 唐雪明[1] LI Peng;ZHANG Lin;YE Ji-Ni;HE Shi-Yao;JIA Jun-Wei;PAN Ai-Hu;TANG Xue-Ming(Biotechnology Research Institute,Shanghai Academy of Agricultural Sciences/Shanghai Key Laboratory of Agricultural Genetics and Breeding,Shanghai 201106,China;School of Life Science,Fudan University,Shanghai 200433,China;Laiwu Vocational and Technical College,Laiwu 200433,Shandong,China;School of Life Science,Taizhou University,Taizhou 318000,Zhejiang,China)

机构地区：[1]上海市农业科学院生物技术研究所/上海市农业遗传育种重点实验室,上海201106 [2]复旦大学生命科学学院,上海200433 [3]莱芜职业技术学院,山东莱芜271100 [4]台州学院生命科学学院,浙江台州318000

出　　处：《作物学报》2018年第7期949-955,共7页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31500461);上海市科技兴农重点攻关项目[沪农科攻字(2015)第4-3];上海市农业科学院卓越团队项目[农科创2017(B-07)];上海市农业转基因生物安全监管专项资助~~

摘　　要：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p M12,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R2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。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,R2 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.

关 键 词：品系特异性 定性PCR 定量PCR M12 

分 类 号：S511[农业科学—作物学]