Chernysheva O N, Sorokina E Iu
Vopr Pitan. 2013;82(3):53-60.
Foodstuffs made by modern biotechnology are requiring for special control. Analytical methods used for these purposes are being constantly perfected. When choosing a strategy for the analysis, several factors have to be assessed: specificity, sensitivity, practically of the method and time efficiency. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. Protein detection methods are based mainly on ELISA. The specific detection of a novel protein synthesized by gene introduced during transformation constitutes an alternative approach for the identification of GMO. The genetic modification is not always specifically directed at the production of a novel protein and does not always result in protein expression levels sufficient for detection purposes. In addition, some proteins may be expressed only in specific parts of the plant or expressed at different levels in distinct parts of plant. As DNA is a rather stable molecule relative to proteins, it is preferred target for any kind of sample. These methods are more sensitive and specific than protein detection methods. PCR-based test can be categorized into several levels of specificity. The least specific methods are commonly called "screening methods" and relate to target DNA elements, such as promoters and terminators that are present in many different GMOs. For routine screening purpose regulatory elements 35S promoter, derived from the Cauliflower Mosaic Virus and the NOS terminator, derived from the nopaline synthase gene of Agrobacterium tumefaciens, are used as target sequences. The second level is "gene-specific methods". These methods target a part of the DNA harbouring the active gene associated with the specific genetic modification. The highest specificity is seen when the target is the unique junction found at the integration locus between the inserted DNA and the recipient genome. These are called "event-specific methods". For a quantitative assessment of GMO the most promising is PCR with the detection of results on the real time-scale. This method has several advantages such as high sensitivity and specificity, little time of analyses, which are conducted in a closed test-tube without contamination environment of PCR products. The microarray technologies have been developed in the past few years. The main principle of the micro-array technology is miniaturization. Methods can be performed on a much larger scale in much smaller volumes.
现代生物技术生产的食品需要进行特殊管控。用于这些目的的分析方法也在不断完善。在选择分析策略时,必须评估几个因素:特异性、灵敏度、方法实用性和时间效率。迄今为止,转基因生物检测方法主要基于转基因生物中插入的DNA序列和新产生的蛋白质。蛋白质检测方法主要基于酶联免疫吸附测定法(ELISA)。对转化过程中导入基因合成的新型蛋白质进行特异性检测是鉴定转基因生物的另一种方法。基因改造并不总是专门针对新型蛋白质的生产,也不总是能产生足以用于检测目的的蛋白质表达水平。此外,一些蛋白质可能只在植物的特定部位表达,或者在植物的不同部位以不同水平表达。由于相对于蛋白质而言,DNA是一种相当稳定的分子,因此它是任何类型样品的首选靶标。这些方法比蛋白质检测方法更灵敏、更具特异性。基于聚合酶链反应(PCR)的检测可分为几个特异性水平。特异性最低的方法通常称为“筛查方法”,涉及靶标DNA元件,如存在于许多不同转基因生物中的启动子和终止子。出于常规筛查目的,源自花椰菜花叶病毒的35S启动子和源自根癌农杆菌胭脂碱合成酶基因的NOS终止子等调控元件被用作靶标序列。第二个水平是“基因特异性方法”。这些方法针对的是包含与特定基因改造相关的活性基因的DNA片段。当靶标是插入的DNA与受体基因组之间整合位点处发现的独特连接时,特异性最高。这些被称为“事件特异性方法”。对于转基因生物的定量评估,最有前景的是实时检测结果的PCR。该方法具有几个优点,如高灵敏度和特异性、分析时间短,且在封闭的试管中进行,不会污染PCR产物环境。微阵列技术在过去几年中得到了发展。微阵列技术的主要原理是小型化。可以在小得多的体积内进行更大规模的操作。