Takabatake Reona, Takashima Kaori, Kurashima Takeyo, Mano Junichi, Furui Satoshi, Kitta Kazumi, Koiwa Tomohiro, Akiyama Hiroshi, Teshima Reiko, Futo Satoshi, Minegishi Yasutaka
National Agriculture and Food Research Organization, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
J AOAC Int. 2013 Mar-Apr;96(2):346-52. doi: 10.5740/jaoacint.12-141.
Qualitative PCR methods for the genetically modified (GM) maize events MON810, Bt11, and GA21, and the 35S promoter (P35S) region of the cauliflower mosaic virus (CaMV) were evaluated in an interlaboratory study. Real-time PCR-based quantitative methods for these GM events using the same primer pairs had already been validated in previous studies. Fifteen laboratories in Japan participated in this interlaboratory study. Each participant extracted DNA from blind samples, performed qualitative PCR assays, and then detected the PCR products with agarose gel electrophoresis. The specificity, sensitivity, and false-negative and false-positive rates of these methods were determined with different concentrations of GM mixing samples. LODs of these methods for MON810, Bt11, GA21, and the P35S segment calculated as the amount of MON810 were 0.2, 0.2, 0.1, and 0.2% or less, respectively, indicating that the LODs of MON810, Bt11, and P35S were lower than 10 copies, and the LOD of GA21 was lower than 25 copies of maize haploid genome. The current study demonstrated that the qualitative methods would be fit for the detection and identification of these GM maize events and the P35S segment.
在一项实验室间研究中，对转基因（GM）玉米事件MON810、Bt11和GA21以及花椰菜花叶病毒（CaMV）的35S启动子（P35S）区域的定性PCR方法进行了评估。此前的研究已经验证了使用相同引物对的基于实时PCR的这些转基因事件定量方法。日本的15个实验室参与了这项实验室间研究。每个参与者从盲样中提取DNA，进行定性PCR分析，然后用琼脂糖凝胶电泳检测PCR产物。使用不同浓度的转基因混合样品确定了这些方法的特异性、灵敏度以及假阴性和假阳性率。以MON810的量计算，这些方法对MON810、Bt11、GA21和P35S片段的检测限分别为0.2%、0.2%、0.1%和0.2%或更低，这表明MON810、Bt11和P35S的检测限低于10拷贝，GA21的检测限低于玉米单倍体基因组的25拷贝。当前研究表明，这些定性方法适用于检测和鉴定这些转基因玉米事件及P35S片段。
