Development and application of DNA analytical methods for the detection of GMOs in food
Development and application of DNA analytical methods for the detection of GMOs in food
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DOI：
10.1016/s0956-7135(99)00081-x
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发表时间：
1999-12-01
期刊：
影响因子：
6
通讯作者：
Meyer, R
中科院分区：
文献类型：
--
作者：
Meyer, R
通过聚合酶链反应（PCR）直接检测食品中重组DNA的原理如下三个主要步骤：DNA提取、PCR扩增和PCR产物验证。适用于从同质、异质、低DNA含量基质中分离基因组DNA的方法讨论了基于经典方案和/或与市售提取试剂盒的组合的凝胶化材料（例如卵磷脂）、凝胶化材料（例如淀粉）、衍生物和成品。各种因素都有助于DNA的降解，例如由于长时间的热处理，核酸酶活性和在低pH值下增加的脱嘌呤和水解而引起的水解。术语“DNA质量”被定义为DNA的降解程度（高度加工食品中的片段大小小于400 bp）以及是否存在PCR的有效抑制剂，因此是一个关键标准。一般来说，在高度热处理的食品、水解植物蛋白（如酱料）、纯化卵磷脂、淀粉衍生物（如麦芽糊精、葡萄糖浆）和确定的化学物质（如精制豆油）中检测不到DNA。如果靶基因或转基因DNA片段的核苷酸序列已知，则可以合成特异性引物并扩增rDNA片段。检测限在20 pg-10 ng靶DNA和0.0001-1%质量分数的GMO范围内。扩增产物然后通过琼脂糖凝胶电泳分离，并通过与DNA分子量标记物比较来估计预期的片段大小。有几种方法用于验证PCR结果，它们在可靠性，精度和成本方面各不相同。它们包括通过限制性核酸内切酶特异性切割扩增产物，或者更耗时但也更特异性地将分离的PCR产物转移到膜上（Southern印迹），然后与靶序列特异性的DNA探针杂交。或者，可以通过直接测序来验证PCR产物。巢式PCR检测结合了高特异性和灵敏度，可用于筛选35 S启动子、NOS、终止子和其他标记基因的方法，这些标记基因用于广泛的转基因生物，可用于特异性检测批准的产品，如FlavrSavr（TM）番茄、Roundup Ready（TM）大豆、Bt-maize 176，以及用于马铃薯和具有模型特征的转基因微生物的官方验证方法。分析新转基因产品的方法正在通过实验室间测试进行验证，新技术正在开发中（例如，EC项目：DMIF-GEN）。然而，由于缺乏转基因参考材料以及迄今为止只能从供应商处获得的特定序列信息，这些努力可能会受到阻碍。(C)1999爱思唯尔科技有限公司。保留所有权利。
The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term "DNA quality" is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg-10 ng target DNA and 0.0001-1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.Methods for the screening of 35S-promoter, NOS;terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr(TM) tomatoes, Roundup Ready(TM) Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e,g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers. (C) 1999 Elsevier Science Ltd. All rights reserved.