State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
Abstract：Rice (Oryza sativa) G6H1 is a new transgenic rice line that has the feature of insect-resistant and herbicide-tolerant. It is developed by Chinese scientists with independent intellectual property rights. The previous studies showed that G6H1 had good performance of insect and glyphosate resistance, and it met the requirement of application level for insect-resistant and herbicide-tolerant, and had a good and broad application prospect. At present, there was lack of methods for accurate quantitative detection of G6H1. In this study, based on the droplet digital PCR (ddPCR) platform, a duplex ddPCR accurate quantification method for G6H1 was established. First, the specificity of primer and probe designed for the G6H1 event was verified by qRT-PCR. The fluorescent signal was not observed in other transgenic crops and non-transgenic crops and only was observed in G6H1 event, these results indicated that designed primer and probe had high specificity for G6H1; then, the suitable different rice endogenous genes for duplex ddPCR detection method were screened and selected, the sucrose phosphate synthase gene (SPS) with 122 length was chosen as the candidate endogenous gene in this study. Moreover, the concentration and annealing temperature of the primer and probe for duplex ddPCR were optimized. The final concentration of primers/probes as 0.5/0.25 μmol/L combined with 58 ℃ annealing temperature were determined as the final optimal condition for the duplex ddPCR. The established duplex ddPCR method for analysis of transgenic rice G6H1 showed that the limit of detection for endogenous and exogenous genes reached 3 copies, and the limit of quantification of endogenous and exogenous genes was 25 copies, and the relative standard deviation (RSD) of quantitative results was less than 25%. Finally, the quantitative results of samples content of 5%, 1%, and 0.5% of transgenic rice G6H1 were compared using simplex qRT-PCR, simplex ddPCR, and duplex ddPCR. All the results showed that the established duplex ddPCR method had good specificity, high sensitivity, accuracy and reliability, and was suitable for precise quantitative analysis of transgenic rice G6H1 event. The establishment of this method provided an alternative method for quantitative detection of transgenic rice G6H1, and improved the detection technology system for transgenic rice components of China. Meanwhile, the established method also can work as new technical reserves for the safefy threshold regulation of genetically modified agricultural organisms.
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