Li Jun, Zhang Li, Li Liang, Li Xiaying, Zhang Xiujie, Zhai Shanshan, Gao Hongfei, Li Yunjing, Wu Gang, Wu Yuhua
Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China.
School of Life Science, South-Central University for Nationalities, Wuhan 430074, China.
ACS Omega. 2020 Aug 18;5(34):21602-21609. doi: 10.1021/acsomega.0c02274. eCollection 2020 Sep 1.
The application of certified reference materials (CRMs) to genetically modified organism (GMO) detection is essential for guaranteeing the accuracy, comparability, and traceability of quantitative results over time and among laboratories. Clean leaves from GM rice Kefeng 6 were used as raw materials to develop a batch of genomic DNA (gDNA) CRMs. The optimized KF6/ duplex digital PCR was used for collaborative characterization of Kefeng 6 gDNA CRMs by eight qualified laboratories; this batch of gDNA CRMs was certified for two property values, namely, copy number ratio and copy number concentration, which were 1.03 ± 0.04 and (1.60 ± 0.11) × 10 copies/μL, respectively. The gDNA CRMs displayed good between-vial homogeneity when the minimum sample intake of 2 μL was taken into account. Stability studies indicated that the gDNA CRMs should be transported below 25 °C, and cold chain transport was recommended. Shelf life was assessed to be at least 12 months, and when using gDNA CRMs, freeze-thaw should not exceed 10 cycles. Compared to the available gDNA CRMs in the market, this batch of gDNA CRMs has accurate property values with combined uncertainties, providing user-friendly calibrators for GM rice Kefeng 6 inspection and monitoring. The development and characterization of Kefeng 6 gDNA CRMs contribute to the establishment of a copy number-based reference system for GMO detection.
标准物质（CRM）在转基因生物（GMO）检测中的应用对于确保定量结果随时间推移以及在不同实验室之间的准确性、可比性和可追溯性至关重要。以转基因水稻克螟稻6号的干净叶片为原料，制备了一批基因组DNA（gDNA）标准物质。采用优化后的KF6/双链数字PCR方法，由八个合格实验室对克螟稻6号gDNA标准物质进行协同特性分析；这批gDNA标准物质被认证了两个特性值，即拷贝数比和拷贝数浓度，分别为1.03±0.04和(1.60±0.11)×10拷贝/μL。当考虑最小进样量为2μL时，gDNA标准物质在瓶间显示出良好的均匀性。稳定性研究表明，gDNA标准物质应在25℃以下运输，建议采用冷链运输。评估其保质期至少为12个月，使用gDNA标准物质时，冻融次数不应超过10次。与市场上现有的gDNA标准物质相比，这批gDNA标准物质具有准确的特性值和合成不确定度，为转基因水稻克螟稻6号的检测和监测提供了用户友好型校准物。克螟稻6号gDNA标准物质的研制和特性分析有助于建立基于拷贝数的转基因生物检测参考体系。
