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A gene-edited transgenic crop editing site-specific PCR method and its application

A gene editing and gene technology, applied in biochemical equipment and methods, microbial assay/inspection, etc., can solve problems such as time-consuming and laborious, inability to meet precise identification and accurate quantitative detection, and inability to identify specific editing sites. , to achieve good specificity and sensitivity

Active Publication Date: 2022-04-22
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the sequencing method, the above methods can only qualitatively detect whether the target gene in the genetically modified product has been edited, but cannot identify the specific editing site, and cannot meet the needs of precise identification and quantitative detection of genetically modified products in the safety supervision of genetically modified organisms ; Although the sequencing method can identify specific editing sites through sequence analysis, this method is relatively time-consuming and laborious, and cannot meet the needs of accurate and quantitative detection of genetically modified products in the safety supervision of genetically modified organisms

Method used

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  • A gene-edited transgenic crop editing site-specific PCR method and its application
  • A gene-edited transgenic crop editing site-specific PCR method and its application
  • A gene-edited transgenic crop editing site-specific PCR method and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Detection of Cao gene edited transgenic rice

[0114] 1. Preparation

[0115] Genomic DNA was extracted from wild-type rice and gene-edited transgenic rice using a DNA extraction kit. Genomic DNA of homozygous gene-edited transgenic rice was serially diluted, and 4-5 concentration gradients were configured as standard samples for fluorescent quantitative PCR.

[0116] 2. Detection operation of Cao gene-edited transgenic rice

[0117] The Cao gene universal primer pair CAO-F / CAO-R was combined with editing site-specific probes, respectively. Specific identification and quantification of Cao gene wild-type rice with primer-probe combination CAO-F / CAO-R / CAO-W-P; specific identification and quantification of Cao with primer-probe combination CAO-F / CAO-R / CAO-E1-P Gene-edited transgenic rice Cao-E1; specific identification and quantification of Cao gene-edited transgenic rice Cao-E2 with primer-probe combination CAO-F / CAO-R / CAO-E2-P; primer-probe combination CAO-...

Embodiment 2

[0128] Example 2 Detection of SP1 Gene Edited Transgenic Rice

[0129] 1. Preparation

[0130] Genomic DNA was extracted from wild-type rice and gene-edited transgenic rice using a DNA extraction kit. Genomic DNA of homozygous gene-edited transgenic rice was serially diluted, and 4-5 concentration gradients were configured as standard samples for fluorescent quantitative PCR.

[0131] 2. Detection operation of SP1 gene edited transgenic rice

[0132] The SP1 gene universal primer pair SP-F / SP-R was combined with the editing site-specific probe respectively. Specific identification and quantification of wild-type rice with the SP1 gene using the primer-probe combination SP-F / SP-R / SP-W-P; specific identification and quantification of SP1 using the primer-probe combination SP-F / SP-R / SP-E1-P Gene-edited transgenic rice SP-E1; Specific identification and quantification of SP1 gene-edited transgenic rice SP-E2 with primer-probe combination SP-F / SP-R / SP-E2-P; Primer-probe combinat...

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Abstract

The invention discloses a gene editing transgenic crop editing site-specific PCR method and its application, and specifically discloses that the detection method includes the following steps. 1) Collect sequences: collect the wild-type nucleotide sequences of target genes and gene-edited nucleotide sequences in gene-edited crops; (2) Design primers / probes: design wild-type nucleotide sequences on both sides of the edited sequence based on the above-mentioned nucleotide sequences. (3) PCR amplification: Using the genomic DNA of gene-edited crops as a template, PCR amplification detection was performed using primer-probe combinations. The invention utilizes universal primers and editing site-specific TaqMan probes, can specifically identify and accurately quantify gene-edited crops, and is suitable for identification and quantitative detection of gene-edited crops.

Description

technical field [0001] The invention relates to a DNA detection method in the field of biotechnology, in particular to an editing site-specific TaqMan probe PCR method for gene-editing transgenic crops, which is used for qualitative and quantitative detection of editing site-specificity of gene-editing transgenic crops. Background technique [0002] As a new breeding technology that has emerged in recent years, gene editing technology can efficiently, precisely and specifically modify plant genomes, and is widely used in the field of plants. For example, DuPont Pioneer Company uses gene editing technology to knock out the Wx1 gene to breed waxy corn, Dow AgroSciences uses ZFN technology to breed a genome edited corn, herbicide-resistant transgenic rapeseed, anti-browning mushrooms, CRISPR / Cas9 Drought-resistant and high-yielding gene-edited maize obtained by technically editing the ARGOS8 promoter region, etc. [0003] Genome editing technology can perform modification oper...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/6851
CPCC12Q1/6895C12Q1/686C12Q1/6851C12Q2600/13C12Q2561/101C12Q2531/113C12Q2545/101
Inventor 武玉花张洪文李俊赵圣博闫晓红高鸿飞李允静吴刚
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI