Hernández Marta, Esteve Teresa, Pla Maria
Molecular Genetics Department, Institut de Biologia Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas, Jordi Girona Salgado 18-26, 08034 Barcelona, Spain.
J Agric Food Chem. 2005 Sep 7;53(18):7003-9. doi: 10.1021/jf050797j.
Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (Hordeum vulgare), rice (Oryza sativa), sunflower (Helianthus annuus), and wheat (Triticum aestivum) are very common among the ingredients of many processed food products; therefore the development of specific assays for their specific detection and quantification are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling/legal, and consumers' information purposes. We report here the development of four independent real-time polymerase chain reaction (PCR) assays suitable for identification and quantification of four plant species (barley, rice, sunflower, and wheat). These assays target gamma-hordein, gos9, helianthinin, and acetyl-CoA carboxylase sequences, respectively, and were able to specifically detect and quantify DNA from the target plant species. In addition, the simultaneous amplification of RALyase allowed bread from durum wheat to be distinguished. Limits of detection were 1 genome copy for barley, sunflower, and wheat and 3.3 copies for rice real-time PCR systems, whereas limits of quantification were 10 genome copies for barley, sunflower, or wheat and approximately 100 haploid genomes for rice real-time PCR systems. Real-time PCR cycling conditions of the four assays were stated as standard to facilitate their use in routine laboratory analyses. The assays were finally adapted to conventional PCR for detection purposes, with the exception of the wheat assay, which detects rye simultaneously with similar sensitivity in an agarose gel.
质量保证是食品工业中的一个主要问题。食品成分的真实性及其可追溯性是消费者和当局所要求的。大麦（Hordeum vulgare）、水稻（Oryza sativa）、向日葵（Helianthus annuus）和小麦（Triticum aestivum）等植物物种在许多加工食品的成分中非常常见；因此，需要开发针对它们进行特异性检测和定量的特定检测方法。此外，越来越多植物物种的转基因品系的生产和贸易带来了在研究、环境风险评估、标签/法律以及消费者信息方面进行管控的需求。我们在此报告了四种独立的实时聚合酶链反应（PCR）检测方法的开发，这些方法适用于鉴定和定量四种植物物种（大麦、水稻、向日葵和小麦）。这些检测方法分别靶向γ-醇溶蛋白、gos9、向日葵蛋白和乙酰辅酶A羧化酶序列，并且能够特异性地检测和定量目标植物物种的DNA。此外，RALyase的同时扩增使得硬粒小麦制成的面包能够被区分出来。大麦、向日葵和小麦实时PCR系统的检测限为1个基因组拷贝，水稻实时PCR系统的检测限为3.3个拷贝，而定量限方面，大麦、向日葵或小麦实时PCR系统为10个基因组拷贝，水稻实时PCR系统约为100个单倍体基因组。这四种检测方法的实时PCR循环条件作为标准给出，以方便它们在常规实验室分析中的使用。除了小麦检测方法外，这些检测方法最终都适用于传统PCR用于检测目的，小麦检测方法在琼脂糖凝胶中能够以相似的灵敏度同时检测黑麦。
