Endogenous reference genes (ERGs) provide vital information regarding genetically modified organisms (GMOs). The successful detection of ERGs can identity GMOs and the source of genes, verify stability and reliability of the detection system, and calculate the level of genetically modified (GM) ingredients in mixtures. The reported ERGs in rice include sucrose-phosphate synthase (SPS), phospholipase D (PLD), RBE4 and rice root-specific GOS9 genes. Based on the characteristics of ERGs, a new ERG gene, phosphoenolpyruvate carboxylase (PEPC), was selected, and further compared with the four existing genes. A total of 18 rice varieties and 29 non-rice crops were used to verify the interspecies specificity, intraspecies consistency, sensitivity, stability and reliability of these five ERGs using qualitative and quantitative PCR. Qualitative detection indicated that SPS and PEPC displayed sufficient specificity, and the detection sensitivity was 0.05% and 0.005%, respectively. Although the specificity of both RBE4 and GOS9 were adequate, the amplicons were small and easily confused with primer dimers. Non-specific amplification of the PLD gene was present in maize and potato. Real-time quantitative PCR detection indicated that PLD, SPS and PEPC displayed good specificity, with R² of the standard curve greater than 0.98, while the amplification efficiency ranged between 90% and 110%. Both the detection sensitivities of PLD and PEPC were five copies and that of SPS was ten copies. RBE4 showed typical amplification in maize, beet and Arabidopsis, while GOS9 was found in maize, tobacco and oats. PEPC exhibited excellent detection sensitivity and species specificity, which made it a potentially useful application in GM-rice supervision and administration. Additionally, SPS and PLD are also suitable for GM-rice detection. This study effectively established a foundation for GMO detection, which not only provides vital technical support for GMO identification, but also is of great significance for enhancing the comparability of detection results, and the standardization of ERG testing in GM-rice.

内源参考基因（ERG）提供了有关转基因生物（GMO）的重要信息。成功检测ERG可以鉴定GMO和基因来源，验证检测系统的稳定性和可靠性，并计算混合物中转基因（GM）成分的水平。水稻中报道的ERGs包括蔗糖磷酸合酶（SPS），磷脂酶D（PLD），RBE4和水稻根特异性GOS9基因。根据ERGs的特征，一个新的ERG基因，磷酸烯醇丙酮酸羧化酶（PEPC）），然后将其与现有的四个基因进行比较。通过定性和定量PCR，总共使用了18个水稻品种和29种非水稻作物来验证这五个ERG的种间特异性，种内一致性，敏感性，稳定性和可靠性。定性检测表明SPS和PEPC具有足够的特异性，检测灵敏度分别为0.05％和0.005％。尽管RBE4和GOS9的特异性都足够，但扩增子很小，容易与引物二聚体混淆。在玉米和马铃薯中存在PLD基因的非特异性扩增。实时定量PCR检测表明PLD，SPS和PEPC表现出良好的特异性，标准曲线的R ²大于0.98，而扩增效率在90％至110％之间。PLD和PEPC的检测灵敏度均为5个拷贝，SPS的检测灵敏度为10个拷贝。RBE4在玉米，甜菜和拟南芥中显示出典型的扩增，而GOS9在玉米，烟草和燕麦中发现。PEPC表现出优异的检测灵敏度和物种特异性，这使其在转基因大米的监督管理中具有潜在的实用性。此外，SPS和PLD也适用于GM大米检测。这项研究有效地为转基因生物的检测奠定了基础，不仅为转基因生物的鉴定提供了重要的技术支持，而且对于增强检测结果的可比性以及在转基因大米中ERG检测的标准化具有重要意义。