关键词: 耐除草剂大豆, 种子, 二重实时荧光PCR, 转化体纯度

Abstract:

【Objective】 Production and application safety certificates have been successively granted to herbicide-tolerant soybean, including SHZD3201, DBN9004, and ZH10-6. The objective of this study is to establish duplex real-time fluorescence PCR detection methods for these three events, which are applied to the accurate and rapid identification of the purity of soybean transgenic event seeds, and to provide technical support for the quality and safety supervision of soybean transgenic varieties seeds.【Method】 Three event-specific real-time PCR methods for herbicide-tolerant soybean: SHZD3201, DBN9004, ZH10-6, and four soybean-specific real-time PCR methods targeting the Lectin were collected. Through the comparison of ΔCt values, the most effective soybean reference gene detection approach for duplex real-time fluorescence PCR was chosen. The study further adjusted the concentration of primers and probes for both events and Lectin, optimizing the reaction system of duplex real-time fluorescence PCR. The duplex real-time fluorescence PCR methods underwent specificity testing using diverse types of samples. The limit of detection (LOD) was assessed using range of gradient samples containing 2 000, 200, 20, 10, 5, and 1 copies. Using one-step extraction solution for rapid grinding of individual seeds, the diluted crude extract was directly used for duplex real-time fluorescence PCR amplification, thereby conducting event purity testing of individual seeds for herbicide-tolerant soybeans SHZD3201, DBN9004, and ZH10-6.【Result】 Following the determination of the minimum ΔCt value through testing and calculation, the optimal soybean reference gene for detecting Lectin was selected in duplex real-time fluorescence PCR of SHZD3201, DBN9004, and ZH10-6 events. After optimization, the SHZD3201/Lectin duplex real-time fluorescence PCR reaction system exhibited optimal performance with the primer/probe concentration of 0.3 μmol·L^-1/0.15 μmol·L^-1 for SHZD3201 event and the primer/probe concentration of 0.3 μmol·L^-1/0.15 μmol·L^-1 for Lectin. In the DBN9004/Lectin duplex real-time fluorescence PCR reaction system, the primer/probe concentrations for both the DBN9004 event and Lectin were identical, setting at 0.4 μmol·L^-1 for the prime and 0.2 μmol·L^-1 for the probe. In the duplex real-time fluorescence PCR reaction system of ZH10-6/Lectin, the primer/probe concentration of ZH10-6 event was 0.4 μmol·L^-1/0.2 μmol·L^-1, the primer/probe concentration for the Lectin was 0.5 μmol·L^-1/0.25 μmol·L^-1. The three methods had good specificity with the LOD of 10 copies each. Using 100 simulated single-seed samples, after rapid grinding and a 5-fold dilution of the template, the direct duplex real-time fluorescence PCR was used for rapid amplification, and the event purity of transgenic seeds of SHZD3201, DBN9004, and ZH10-6 was successfully detected.【Conclusion】 This study successfully established three duplex real-time fluorescence PCR methods for herbicide-tolerant soybean SHZD3201, DBN9004, and ZH10-6. By combining a rapid DNA extraction method using one-step extraction solution, the research achieved precise and rapid detection of event purity in transgenic soybean seeds.

Key words: herbicide-tolerant soybean, seed, duplex real-time fluorescence PCR, event purity