Holst-Jensen Arne, Rønning Sissel B, Løvseth Astrid, Berdal Knut G
National Veterinary Institute, P.O. Box 8156 Dep., 0033, Oslo, Norway.
Anal Bioanal Chem. 2003 Apr;375(8):985-93. doi: 10.1007/s00216-003-1767-7. Epub 2003 Feb 15.
Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications. The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods.
尽管聚合酶链式反应(PCR)技术存在明显局限性,但其潜在的高灵敏度和特异性解释了为何它一直是大多数对检测转基因生物(GMOs)及其衍生材料感兴趣的分析实验室的首选方法。由于实验室收到用于分析的产品通常经过加工和提纯,目标分析物(如蛋白质或DNA)的质量和数量常常对任何检测方法的灵敏度构成挑战。在目前可用的方法中,PCR方法通常被认为是在常规应用中检测转基因衍生材料最灵敏、最可靠的方法。目标序列基序的选择是控制PCR方法特异性的唯一最重要因素。目标序列通常是修饰基因构建体的一部分,例如启动子、终止子、基因或这些元件中两个元件之间的连接区。然而,这些元件可能源自野生型生物,可能存在于不止一种转基因生物中,并且它们的拷贝数也可能因转基因生物的不同而有所变化。它们甚至可能以类似的方式组合在不止一种转基因生物中。因此,方法的选择应符合目的。最近的进展包括事件特异性方法,这对于鉴定和定量转基因含量特别有用。现在许多国家,包括欧盟国家,都制定了标签阈值。标签计划的成功取决于检测转基因衍生材料的效率。我们将概述目前用于筛选和定量转基因衍生DNA的PCR方法,并讨论它们的适用性和局限性。此外,我们将讨论与确定检测限(LOD)和定量限(LOQ)以及方法验证相关的一些主要挑战。