Development of a certified genomic DNA reference material for detection and quantification of genetically modified rice
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RESEARCH PAPER
Development of a certified genomic DNA reference material for detection and quantification of genetically modified rice KMD Jun Li 1 & Liang Li 2 & Li Zhang 3 & Xiujie Zhang 4 & Xiaying Li 4 & Shanshan Zhai 1 & Hongfei Gao 1 & Yunjing Li 1 & Gang Wu 1 & Yuhua Wu 1 Received: 21 May 2020 / Revised: 9 July 2020 / Accepted: 20 July 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Qualitative and quantitative detection of genetically modified products is inseparable from the application of reference materials (RMs). In this study, a batch of genomic DNA (gDNA) certified reference materials (CRMs) was developed using genetically modified rice Kemingdao (KMD) homozygotes as the raw material. The gDNA CRMs in this batch showed good homogeneity; the minimum sample intake was determined to be 2 μL. The stability study showed that transportation by cold chain is preferable, no significant degradation trend was observed during a 12-month period when storing the gDNA CRMs at 4 °C and − 20 °C, and the number of freeze-thaw cycles cannot exceed 10. The property values of the copy number ratio of transgene and endogenous gene and the copy number concentration for gDNA CRMs were determined by a collaborative characterization of eight laboratories using the duplex KMD/PLD droplet digital PCR (ddPCR) assays. The uncertainty components of characterization, potential between-unit heterogeneity, and potential degradation during long-term storage were combined to estimate the expanded uncertainty of the certified value with a coverage factor k of 2.0. The certified value of copy number ratio for KMD gDNA CRM is 0.99 ± 0.05, and that of copy number concentration is (1.76 ± 0.10) × 105 copies/μL. Compared to the gDNA CRMs in availability, this batch of KMD gDNA CRMs is assigned accurate property values and can be directly used for qualitative and quantitative detection of GMOs as well as evaluation of the parameters of analytical methods with no need of further DNA concentration measurement. Keywords KMD . Genomic DNA . Certified reference materials . Droplet digital PCR . Collaborative characterization Jun Li and Liang Li contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02834-y) contains supplementary material, which is available to authorized users. * Xiujie Zhang [email protected] * Gang Wu [email protected] * Yuhua Wu [email protected] 1
Key Laboratory of Biology and Genetic Improvement of Oil Crops of the Ministry of Agriculture, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, Hubei, China
2
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
3
School of Life Science, South-Central University for Nationalities, Wuhan 430074, Hubei, China
4
Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs P. R. China, Beijing 100025, China
Introduction More and more genetically modified organis
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