National Center for Molecular Characterization of Genetically Modified Organisms, SJTU-Bor Luh Food Safety Center, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
PLoS One. 2013 Sep 30;8(9):e75850. doi: 10.1371/journal.pone.0075850. eCollection 2013.
Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.
在转基因生物（GMO）检测中，正确选择内参基因及其实时定量 PCR 检测方法非常重要。为了找到适合普通小麦（Triticum aestivum L.）DNA 含量或拷贝数定量的内参基因及其实时定量 PCR 检测方法，对之前报道的 4 个小麦内参基因及其实时定量 PCR 检测方法进行了全面评估，以了解其目标基因序列变异情况及其在 37 个普通小麦品系中的实时定量 PCR 性能。在 PKABA1 和 ALMT1 基因中观察到 3 个 SNP，这些 SNP 显著降低了实时 PCR 扩增效率。对普通小麦品系中每个基因的实时定量 PCR 性能进行 GeNorm 分析表明，Waxy-D1 检测法在所有检测品系中具有最低的 M 值和最佳的稳定性。所有结果表明，Waxy-D1 基因及其实时定量 PCR 检测法最适合作为普通小麦 DNA 含量定量的内参基因。经过验证的 Waxy-D1 基因检测法将有助于建立 GM 小麦准确可信的定性和定量 PCR 分析。
