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Specific primer for quantitatively detecting transgenic soybean ZH10-6 and method

A technology for quantitative detection of genetically modified soybeans, which is applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc.

Inactive Publication Date: 2020-11-03
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention overcomes the technical defect that ordinary PCR detection of transgenic soybean ZH10-6 can only be qualitative but not quantitative, and provides a method for quantitative detection of transgenic soybean ZH10-6
The purpose of the present invention is to solve the problem of the vacancy of the method for quantitative detection of transgenic soybean ZH10-6, which has the advantages of simplicity, high sensitivity, strong specificity and stability

Method used

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  • Specific primer for quantitatively detecting transgenic soybean ZH10-6 and method
  • Specific primer for quantitatively detecting transgenic soybean ZH10-6 and method
  • Specific primer for quantitatively detecting transgenic soybean ZH10-6 and method

Examples

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Effect test

Embodiment 1

[0043] (1) Sample 1: A self-made simulated mixed sample of 5% genetically modified herbicide-tolerant soybean ZH10-6. Mixing method: 50g of genetically modified herbicide-tolerant soybean ZH10-6 and 950g of non-transgenic soybean ZH10 were thoroughly mixed.

[0044] (2) Reagents: Premix Ex Taq (Probe qPCR) master mix produced by TaKaRa Company; soybean endogenous Lectin gene and transformant-specific sequence amplification primers synthesized by Shanghai Sangon.

[0045] Transformant-specific sequence primers:

[0046] Forward primer sequence: 5'-CAAATCCTATGGGCATTCTTCC-3'

[0047] Reverse primer sequence: 5'-CTAGAGCAGCTTGAGCTTGGATC-3'

[0048] Probe sequence: FAM-CACCTTCTGGCTCCTTCAAACACTG-BHQ1

[0049] endogenous gene Lectin gene sequence:

[0050] Forward primer sequence: 5'-GCCCTCTACTCCACCCCCCA-3'

[0051] Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'

[0052] Probe sequence: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;

[0053] (3) PCR reaction system: total volume 20...

Embodiment 2

[0067] (1) Sample 2: GM herbicide-tolerant soybean ZH10-6 self-made simulated mixed sample with 3% ingredients, mixing method 30g of genetically modified herbicide-tolerant soybean ZH10-6 and 970g of non-genetically modified soybean ZH10 were thoroughly mixed.

[0068] (2) Reagents: Premix Ex Taq (Probe qPCR) master mix produced by TaKaRa Company; soybean endogenous Lectin gene and transformant-specific sequence amplification primers synthesized by Shanghai Sangon.

[0069] Transformant-specific sequence primers:

[0070] Forward primer sequence: 5'-CAAATCCTATGGGCATTCTTCC-3'

[0071] Reverse primer sequence: 5'-CTAGAGCAGCTTGAGCTTGGATC-3'

[0072] Probe sequence: FAM-CACCTTCTGGCTCCTTCAAACACTG-BHQ1

[0073] endogenous gene Lectin gene sequence:

[0074] Forward primer sequence: 5'-GCCCTCTACTCCACCCCCCA-3'

[0075] Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'

[0076] Probe sequence: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;

[0077] (3) PCR reaction system: total volume...

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Abstract

The invention discloses a specific primer for quantitatively detecting transgenic soybean ZH10-6. A pair of specific primers and a DNA polymerase with strand displacement activity are adopted, a fluorescently labeled probe is added into a PCR reaction system to monitor the PCR reaction process in real time, and nucleic acid is amplified at about 60 DEG C. A standard curve is made by diluting a standard substance, the percentage content of a transgenic herbicide-resistant soybean ZH10-6 transformant sequence is corrected by utilizing an internal reference, and an unknown sample is subjected torelative quantitative detection. For the result identification, whether amplification occurs or not is judged by judging whether an amplification curve is peaked or not and judging the cycle number ofpeaking, and the percentage content of an unknown sample is relatively quantified by utilizing the standard curve made by the standard substance. The detection method disclosed by the invention has the advantages of high specificity, high sensitivity, rapidness, simplicity, convenience and the like, and a feasible method is provided for quantitative detection of the transgenic herbicide-resistantsoybean ZH10-6.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for detecting transgenic products, specifically a method for quantitatively detecting transgenic soybean ZH10-6 based on real-time fluorescent PCR. A method for quantitatively detecting the sequences of transgenic herbicide-tolerant soybean ZH10-6 transformants by gene correction of exogenous species-specific genes. Background technique [0002] According to the latest statistics from ISAAA, an international service organization for agricultural biotechnology applications, in 2018, 26 countries around the world planted 191.7 million ha 2 Genetically modified crops, of which 95.9 million hectares of genetically modified soybeans 2 The planting area ranks first, accounting for 50% of the global planting area of ​​genetically modified crops; in terms of the planting area of ​​a single crop, the application rate of genetically modified soybeans in 2018...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/13C12Q2600/166
Inventor 赵新王一衡刘双尉万聪兰青阔王永
Owner 天津市农业科学院
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