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RPA primer and probe combination for detecting transgenic soybean SHZD32-1 and on-site rapid detection method

A technology of transgenic soybeans and probes, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of restricting constant temperature amplification technology, not being able to meet rapid detection, not being able to connect well, etc., to achieve Broad application prospects, good operability, and the effect of simplifying steps and procedures

Pending Publication Date: 2021-02-12
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

[0005] However, there are currently two main problems restricting the application of constant temperature amplification technology in on-site detection. One is the rapid extraction of nucleic acids, and the other is rapid amplification and result discrimination.
On the one hand, the traditional nucleic acid extraction method is complicated, time-consuming and labor-intensive. Even with the commonly used nucleic acid extraction kits, the extraction time is about 1 hour, and centrifuges and other equipment are required, which cannot meet the needs of rapid on-site detection.
On the other hand, although the constant temperature amplification technology ensures the rapid amplification of nucleic acid, it cannot be well connected with the judgment of the later results. At present, fluorescent dyes are often added after the amplification, which increases the operation steps and increases the aerosol The risk of contamination; there is the use of fluorescence instruments to detect the amplification signal
None of these methods can fully meet the needs of on-site rapid detection

Method used

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  • RPA primer and probe combination for detecting transgenic soybean SHZD32-1 and on-site rapid detection method
  • RPA primer and probe combination for detecting transgenic soybean SHZD32-1 and on-site rapid detection method
  • RPA primer and probe combination for detecting transgenic soybean SHZD32-1 and on-site rapid detection method

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Embodiment

[0050] figure 1 It is a schematic diagram of the on-site rapid detection method for transgenic soybean SHZD32-1 of the present invention, wherein A is the rapid sample extraction process, B is the RPA amplification and result discrimination process, and C is the schematic diagram of the exo probe principle.

[0051] 1.1 Materials and reagents

[0052] 1.1.1 Materials

[0053] Test materials: leaves of transgenic soybean SHZD32-1 were obtained from the transgenic base of the laboratory of Zhejiang Academy of Agricultural Genetically modified soybeans, genetically modified corn (NK603, BT176, T25), genetically modified cotton (LLcotton25, MON15985, GHB119), and genetically modified rapeseed (MS1×RF1, MS1×RF2, OXY235) are all preserved by our laboratory.

[0054] Both the filter membrane and the adsorption membrane were purchased from Hangzhou Laifeng Biotechnology Co., Ltd.

[0055] 1.1.2 Reagents

[0056] RPA constant temperature rapid amplification kits (basic type and flu...

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Abstract

The invention provides an RPA primer and probe combination for detecting transgenic soybean SHZD32-1 and an on-site rapid detection method. RPA primers and a probe are designed for a transformant specific sequence of the transgenic soybean SHZD32-1, a DNA rapid extraction method is combined, a detection result is directly judged through irradiation of a fluorescent lamp, and on-site rapid visual detection of the transgenic soybean SHZD32-1 can be completed within 30 minutes. According to the invention, special instruments are not needed, and rapid and specific detection of nucleic acid under non-experimental conditions (such as a soybean planting base) can be realized.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to an RPA primer and probe combination and an on-site rapid detection method for detecting transgenic soybean SHZD32-1. Background technique [0002] Currently, GM soybeans are the most widely planted GM crops in the world. my country is the country with the largest consumption of genetically modified soybeans in the world. my country's genetically modified soybeans mainly rely on imports. Therefore, it is of great significance to research and develop transgenic soybeans with independent property rights in my country. SHZD32-1 glyphosate-tolerant soybean (that is, herbicide-tolerant soybean SHZD32-1) is a transgenic soybean independently developed by Shanghai Jiaotong University in my country. It uses the Agrobacterium-mediated method to introduce the optimized glyphosate-resistant gene G10-EPSPS. Glyphosate-tolerant soybeans with transgenic G10-EPSPS gene were...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2523/308C12Q2563/107
Inventor 汪小福陈欲徐俊锋陈笑芸彭城徐晓丽魏巍杨蕾
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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