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RPA primer probe combination, kit and detection method for detecting transgenic corn DBN9958

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

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 line-specific identification of transgenic maize DBN9958 using RPA technology

Method used

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  • RPA primer probe combination, kit and detection method for detecting transgenic corn DBN9958
  • RPA primer probe combination, kit and detection method for detecting transgenic corn DBN9958
  • RPA primer probe combination, kit and detection method for detecting transgenic corn DBN9958

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, design and screening of primers and probe combinations

[0032] Primers and probes were designed according to the specific regions of transgenic maize DBN9958 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 individual...

Embodiment 2

[0036] Example 2. Specificity and sensitivity analysis of the detection of transgenic corn DBN9958 using the primers and probe combinations screened in Example 1

[0037] 1. Experimental materials

[0038] 1.1 Plant material

[0039] Genetically modified corn DBN9958, genetically modified corn DBN9958 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, non- Mixed samples of genetically modified soybeans, mixed samples of non-genetically modified cotton, and mixed samples of non-genetically modified rapeseed.

[0040] 1.2 Enzymes and Reagents

[0041] Molecular biology reagents, TwistAmp DNA amplification Exo Kits were purchased from TwistDX Company, TwistAmp DNA amplification Basic Kits were purchased ...

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Abstract

The invention relates to an RPA detection primer, probe combination, kit and detection method of transgenic corn DBN9958. Massive RPA primers are designed according to a connection region of an exogenous insertion DNA sequence and a corn genome, and a primer and probe combination capable of quickly and effectively detecting the component of the transgenic corn DBN9958 is screened out from the RPAprimers. The transgenic corn DBN9958 genome DNA is used as a template, the primer and probe combination is used for RPA amplification and real-time fluorescence detection, and the primer and probe combination has the advantages 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 DBN9958. Background technique [0002] Genetically modified corn DBN9958 is a glyphosate and glufosinate-resistant genetically modified corn variety developed by Beijing Dabeinong Biotechnology Co., Ltd. It is a glyphosate-resistant corn variety obtained by introducing the epsps gene and pat gene into corn by using genetic engineering technology. Transgenic strains of phosphine and glufosinate herbicides. This strain has obtained the agricultural transgenic organism safety certificate (production application) in June 2020, and has broad prospects for industrial application. The reported detection method of transgenic corn DBN9958 mainly uses PCR instrument for routine detection in the laboratory, but PCR-based transgenic detection method requires professional equipment such as P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2563/107C12Q2521/507C12Q2522/101C12Q2537/1376
Inventor 宛煜嵩孟丽霞
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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