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RPA (recombinase polymerase amplification) primer probe combination for detecting transgenic maize G1105E-823C, kit and detection method

A technology of genetically modified corn and a detection kit, which is applied in the field of molecular biology to achieve high sensitivity, good specificity and fast speed

Pending Publication Date: 2020-12-11
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no line-specific identification method for transgenic maize G1105E-823C using RPA technology

Method used

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  • RPA (recombinase polymerase amplification) primer probe combination for detecting transgenic maize G1105E-823C, kit and detection method
  • RPA (recombinase polymerase amplification) primer probe combination for detecting transgenic maize G1105E-823C, kit and detection method
  • RPA (recombinase polymerase amplification) primer probe combination for detecting transgenic maize G1105E-823C, kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1, design and screening of primers and probe combinations

[0033] Primers and probes were designed according to the specific regions of transgenic maize G1105E-823C transformants. When designing the primers, avoid the formation of secondary structures between the front and back primers and the occurrence of repeated sequences between the primers. Select two T bases in the middle and back of the probe, and label each with a fluorescent group (FAM and BHQ1). There is an abasic site (THF) between the two groups, which is recognized and cut by an exonuclease during the reaction, so that the two fluorescent groups are separated to generate a fluorescent signal, and the specific probe can be real-time Monitor the results of the fluorescence detection. The length of the primer is about 35nt. RPA experiments need to design multiple pairs of primers from both ends of the target sequence for optimization and screening. The increase, decrease or substitution of indivi...

Embodiment 2

[0037] Example 2. Specificity and sensitivity analysis of the detection of transgenic maize G1105E-823C using the primers and probe combinations screened in Example 1

[0038] 1. Experimental materials

[0039] 1.1 Plant material

[0040] Genetically modified corn G1105E-823C, genetically modified corn G1105E-823C recipient material, other genetically modified corn mixed samples, genetically modified rice mixed samples, genetically modified soybean mixed samples, genetically modified cotton mixed samples, genetically modified rapeseed mixed samples, non-genetically modified corn mixed samples, non-genetically modified rice Mixed samples, mixed samples of non-GMO soybeans, mixed samples of non-GMO cotton, and mixed samples of non-GMO rapeseed.

[0041] 1.2 Enzymes and Reagents

[0042] Molecular biology reagents, TwistAmp DNA amplification Exo Kits were purchased from TwistDX Company, TwistAmp DNA amplification Basic Kits were purchased from TwistDX Company, and other biochemic...

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Abstract

The invention relates to an RPA (recombinase polymerase amplification) detection primer and probe combination of transgenic maize G1105E-823C, a kit and a detection method. A large number of RPA primers are designed according to a connecting region of an exogenous insertion DNA sequence and a maize genome, and a primer probe combination capable of quickly and effectively detecting the transgenic maize G1105E-823C component is screened out. Transgenic maize G1105E-823C genome DNA is used as a template, the primer probe combination is used for RPA amplification and real-time fluorescence detection, and the detection primer and probe combination, the kit and the detection method have the characteristics of being high in speed, good in specificity and high in sensitivity.

Description

technical field [0001] This application relates to the technical field of molecular biology, in particular, to an RPA primer-probe combination, kit and detection method for detecting transgenic corn G1105E-823C. Background technique [0002] Transgenic corn G1105E-823C is a glyphosate herbicide-resistant transgenic corn variety developed by Beijing ORG Seed Co., Ltd. It is obtained by introducing the G2-aroA gene from Pseudomonas fluorescens into corn by using genetic engineering technology Transgenic lines resistant to glyphosate herbicides. This strain has entered the application stage of biosafety evaluation safety certificate (production application), and has broad industrialization prospects. In the reported detection methods for transgenic corn G1105E-823C, the PCR instrument is mainly used for routine detection in the laboratory. It takes a long time (about 3 to 4 hours) for the detection of transgenes and products, and it is difficult to achieve the purpose of rapi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2600/13C12Q2600/158
Inventor 宛煜嵩孟丽霞
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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