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RPA primer-probe combination, kit and detection method for detecting double resistance 12-6 of transgenic maize

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

Inactive Publication Date: 2021-02-02
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

At present, there is no method for strain-specific identification of transgenic maize double antibody 12-6 using RPA technology

Method used

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  • RPA primer-probe combination, kit and detection method for detecting double resistance 12-6 of transgenic maize
  • RPA primer-probe combination, kit and detection method for detecting double resistance 12-6 of transgenic maize
  • RPA primer-probe combination, kit and detection method for detecting double resistance 12-6 of transgenic maize

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, design and screening of primers and probe combinations

[0030] Primers and probes were designed according to the specific region of the transgenic maize double antibody 12-6 transformant. 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 substitutio...

Embodiment 2

[0034] Example 2. Specificity and sensitivity analysis of the detection of transgenic maize double antibody 12-6 using the primers and probe combinations screened in Example 1

[0035] 1. Experimental materials

[0036] 1.1 Plant material

[0037] Genetically modified corn double-antibody 12-6, genetically modified corn double-antibody 12-6 receptor 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 , mixed samples of non-genetically modified rice, mixed samples of non-genetically modified soybeans, mixed samples of non-genetically modified cotton, and mixed samples of non-genetically modified rapeseed.

[0038] 1.2 Enzymes and Reagents

[0039] Molecular biology reagents, TwistAmp DNA amplification Exo Kits were purchased from TwistDX Company, TwistAmp DN...

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Abstract

The invention discloses a RPA (Recombinase Polymerase Amplification) primer / probe combination, a kit and a detection method for detecting double-resistance 12-6 of transgenic maize. A large number ofRPA primers are designed according to a connection region of a foreign inserted DNA (Deoxyribonucleic Acid) sequence and the maize genome, and a primer-probe combination capable of rapidly and effectively detecting the transgenic maize double resistance 12-6 component is screened out from the RPA primers. Using the genomic DNA of transgenic maize double resistance 12-6 as a template, the primer-probe combination is used for RPA amplification and real-time fluorescence detection, which has the characteristics of fast speed, good specificity and high 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 maize double antibody 12-6. Background technique [0002] The transgenic corn Shuangkang 12-6 is an insect-resistant and glyphosate herbicide-resistant transgenic corn strain developed by Zhejiang University. It is a corn borer-resistant corn borer-resistant gene obtained by introducing cry1Ab / cry2Aj fusion gene and G10evo-epsps gene into corn by genetic engineering technology. and glyphosate-tolerant transgenic lines. The strain has entered the stage of productive testing for safety evaluation of genetically modified organisms, and has broad prospects for industrialization. Among the reported detection methods for the double-antibody 12-6 of transgenic corn, the PCR instrument is mainly used for routine detection in the laboratory, but the PCR-based transgenic detection method requi...

Claims

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Application Information

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