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| Establishment of a Real-time Fluorescence Recombinase Polymerase Amplification for Detection of Transgenic Soybean (Glycine max) MON89788 |
| XIE Shi-Long1,2, WANG Xiao-Fu2*, DING Chen-Lu1, ZHU Xuan1,2, TANG Ting1,2, MA Tong-Fu1, CAI Jian1, XU Jun-Feng2* |
1 Fuyang Normal University, Fuyang 236037, China;
2 State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China |
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Abstract Transgenic soybean (Glycine max) MON89788 is approved for commercial planting earlier. It has a wide range of cultivation with a large circulation of products. In this study, a series of primers combining with a special probe were designed for recombinase polymerase amplification (RPA) based on the event-specific sequence of MON89788. Then a strategy that forward primers and reverse primers mutually amplifying was employed to obtain the optimal primers combination with the highest amplification efficiency. In addition, the RPA reaction conditions, including reaction temperature, the concentration of primers and probe, were selected and optimized. The results indicated that RPA had a wide range of amplification temperature: Among 29.5~43.1 ℃, and the high concentration of probe in the reaction system would affect the amplification efficiency of RPA. Further, the specificity, sensitivity and applicability of the RPA detection system were tested. Then the MON89788 real-time fluorescent RPA (RT-RPA) detection method was established. The detection method was specific, and the absolute limit of detection (aLOD) of MON89788 could reach 40 copies, the relative limit of detection (rLOD) was 0.05%. Furthermore, for real samples detection, the RT-RPA detection was completed within 10 min at 39 ℃, which was 0.07~0.13 times of quantitative real-time PCR (qRT-PCR) detection time. This isothermal and rapid detection method provides new technical support for the rapid detection of genetically modified components, and is expected to be used for rapid on-site detection of genetically modified components.
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Received: 28 November 2018
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Corresponding Authors:
yywxf1981@163.com; njjfxu@163.com
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