Selection of Suitable DNA Extraction Methods for Genetically Modified Maize 3272, and Development and Evaluation of an Event-Specific Quantitative PCR Method for 3272

摘要

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize, 3272. We first attempted to obtain genome DNA from this maize using a DNeasy Plant Maxi kit and a DNeasy Plant Mini kit, which have been widely utilized in our previous studies, but DNA extraction yields from 3272 were markedly lower than those from non-GM maize seeds. However, lowering of DNA extraction yields was not observed with GM quicker or Genomic-tip 20/G. We chose GM quicker for evaluation of the quantitative method. We prepared a standard plasmid for 3272 quantification. The conversion factor (Cf), which is required to calculate the amount of a genetically modified organism (GMO), was experimentally determined for two real-time PCR instruments, the Applied Biosystems 7900HT (the ABI 7900) and the Applied Biosystems 7500 (the ABI7500). The determined Cf values were 0.60 and 0.59 for the ABI 7900 and the ABI 7500, respectively. To evaluate the developed method, a blind test was conducted as part of an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSDr). The determined values were similar to those in our previous validation studies. The limit of quantitation for the method was estimated to be 0.5% or less, and we concluded that the developed method would be suitable and practical for detection and quantification of 3272.%遺伝子組換えトウモロコシ3272系統のリアルタイムPCRを用いた系統特異的定量分析法を開発した.DNA抽出に関しては,DNeasy Plant Maxi kitおよびDNeasy Plant Mini kitを用いたところ,非組換え種子に比べて抽出DNA量が有意に低かったif,GM quickerおよびGenomic-tip 20/Gを用いたところ,このような差は見られなかった.GM quickerで抽出精製したDNAを用いて試験室間共同試験を実施し,混入率算出の際に必要となる係数である内標比を決定した.さらに,ブラインド定量試験を実施したところ,本定量分析法の定量下限値は0.5%程度と見積もられた.以上の結果から,本分析法が,実際の検査に適用可能であることが示された.