Grain Research Laboratory, Canadian Grain Commission, 1404-303 Main Street, Winnipeg, MB R3C 3G8, Canada.
Anal Bioanal Chem. 2010 Mar;396(6):1977-90. doi: 10.1007/s00216-009-3150-9. Epub 2009 Sep 30.
Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.
美国、加拿大和其他主要的粮食出口国正在种植生物技术衍生的油菜、棉花、玉米和大豆品种。尽管用于生产生物技术衍生作物的农田面积继续增加，但人们仍然担心可能会出现意想不到的后果，这为欧盟和大多数粮食进口国提供了监管这些作物的理由。欧盟的立法要求对谷物/油籽、食品和饲料产品进行可追溯性，并在分析样本中证明存在 0.9%w/w 的转基因特征阈值时进行标记。GE 含量通常通过定量 PCR（qPCR）确定，植物基因组 DNA 为该过程的初始步骤提供模板。存在大量适用于 qPCR 应用的 DNA 提取方法。实施用于检测转基因特征的标准化方法对于促进谷物营销是必要的。国际标准化组织（ISO）标准草案 21571 确定了基于洗涤剂的方法和广泛用于 DNA 提取的商业试剂盒，但也表明根据样品基质可能需要进行调整。本综述评估了各种市售 DNA 提取试剂盒的优缺点，以及对已发表的十六烷基三甲基溴化铵方法的修改。抑制剂是 qPCR 中有效扩增的主要障碍。讨论了 PCR 抑制剂的类型和最小化抑制的技术。最后，对于 qPCR 应用中的 DNA 准确定量并不简单。当应用特定的 DNA 定量方法时，许多混杂因素会导致分析测量结果存在差异，并且不同的方法并不总是在相同的 DNA 样本上提供一致的结果。考虑这些差异如何影响 qPCR 中的测量不确定度。
