Jiang Lingxi, Yang Litao, Zhang Haibo, Guo Jinchao, Mazzara Marco, Van den Eede Guy, Zhang Dabing
GMO Detection Laboratory, Shanghai Jiao Tong University (SJTU)-Bor Luh Food Safety Center, School of Life Science and Biotechnology, SJTU, 800 Dongchuan Road, Shanghai 200240, People's Republic of China.
J Agric Food Chem. 2009 May 13;57(9):3525-32. doi: 10.1021/jf803166p.
One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates.
在之前的一项研究中，已证实一个水稻（Oryza sativa）基因——蔗糖磷酸合酶（SPS），是转基因水稻检测中合适的内参基因。本文报道了一项国际合作的环试结果，用于验证SPS基因作为内参基因及其优化的定性和定量聚合酶链反应（PCR）系统。来自七个国家的总共12个转基因生物（GMO）检测实验室参与了环试并反馈了结果。验证结果通过对10种植物基因组DNA进行测试，证实了该方法的物种特异性、低异质性以及水稻SPS基因在7个籼稻品种和5个粳稻品种中的稳定单拷贝数。SPS定性PCR检测方法的检测限（LOD）验证为0.1%，相当于约230个单倍体水稻基因组DNA拷贝，而定量PCR系统的定量限（LOQ）约为23个单倍体水稻基因组DNA拷贝，具有可接受的PCR效率和线性。此外，八个盲样的测试值与真实值之间的偏差范围为5.22%至26.53%。因此，我们认为SPS基因适用于转基因水稻及其衍生物的鉴定和定量的内参基因。
