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Method for accurately and quantitatively detecting transgenic herbicide-resistant soybean ZH10-6

A transgenic soybean and herbicide-tolerant technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of vacancy in the method of transgenic soybean ZH10-6, which can only be qualitative but not quantitative

Pending Publication Date: 2020-12-11
天津市农业科学院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention overcomes the defect that the qualitative PCR detection technology of transgenic soybean ZH10-6 can only be qualitative but not quantitative, and provides a method for accurate and 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 fast, efficient, accurate quantitative detection, etc.

Method used

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  • Method for accurately and quantitatively detecting transgenic herbicide-resistant soybean ZH10-6
  • Method for accurately and quantitatively detecting transgenic herbicide-resistant soybean ZH10-6
  • Method for accurately and quantitatively detecting transgenic herbicide-resistant soybean ZH10-6

Examples

Experimental program
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Effect test

Embodiment 1

[0047] (1) Sample 1: A self-made simulated mixed sample with 50% specific sequence components of the transgenic herbicide-tolerant soybean ZH10-6 transformant. The mixing method is 50g of transgenic soybean ZH10-6 powder + 50g of non-transgenic soybean acceptor ZH10 powder.

[0048] (2) Reagent: BIO-RAD ddPCR Supermix for Probes master mix; endogenous soybean synthesized by Shanghai Sangong Lectin Gene and transformant specific sequence amplification primers.

[0049] Transformant-specific sequences:

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

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

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

[0053] endogenous gene Lectin gene sequence:

[0054] Forward primer sequence: 5'-GCCCTCTACTCCACCCCCCA-3'

[0055] Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'

[0056] Probe sequence: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;

[0057] (3) PCR reaction system: the total volume is 2...

Embodiment 2

[0069] (1) Sample 2: Soybean samples from three generations of transgenic herbicide-tolerant soybean ZH10-6.

[0070] (2) Reagent: BIO-RAD ddPCR Supermix for Probes master mix; endogenous soybean synthesized by Shanghai Sangong Lectin Gene and transformant specific sequence amplification primers.

[0071] Transformant-specific sequences:

[0072] Forward primer sequence: 5'-GCGGTTTCTTTCAAATCCTATGG-3'

[0073] Reverse primer sequence: 5'-CGTTTCCCGCCTTCAGTTTA-3'

[0074] Probe sequence: FAM-AATGCCACCTTCTGGCTCCTTCAAACAC-BHQ1;

[0075] endogenous gene Lectin gene sequence:

[0076] Forward primer sequence: 5'-GCCCTCTACTCCACCCCCCA-3'

[0077] Reverse primer sequence: 5'-GCCCATCTGCAAGCCTTTTT-3'

[0078] Probe sequence: FAM-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1;

[0079] (3) PCR reaction system: the total volume is 20 μL, including 10 μL of BIO-RAD ddPCR Supermix for Probes, 1 μL of upstream primer (10 μmol / L), 1 μL of downstream primer (10 μmol / L), probe (10 µmol / L) 1 μL, DNA t...

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Abstract

The invention discloses a method for accurately and quantitatively detecting transgenic herbicide-resistant soybean ZH10-6. According to the method, a pair of specific primers, a probe with fluorescent markers, and DNA polymerase with strand displacement activity are used, a PCR system is distributed into reaction units which is small enough through a droplet generator before a PCR reaction, the situation that each reaction unit has only one template molecule is realized, the PCR amplification is performed on nucleic acid at the temperature of about 60 DEG C, and then the Poisson distributionprinciple is used to calculate a target molecule copy number according to a ratio of the number of positive droplets to the number of negative droplets, so that the absolute quantitative analysis is realized, a standard substance is not needed, and the standard curve construction is not needed. The result identification comprises the steps of reading the number of positive droplets and the numberof negative droplets through a droplet reader, and calculating the transgenic content and transformant copy number of a sample according to a ratio of the copy number concentration of an exogenous gene to the copy number concentration of an internal standard gene. The detection method provided by the invention has the advantages of accurate quantification, high specificity, high sensitivity, rapidness, simplicity, convenience and the like.

Description

technical field [0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a precise quantitative detection method for transgenic products, specifically a digital PCR-based precise quantitative detection method for transgenic soybean ZH10-6. By generating microdroplets from the system solution, the The PCR system is allocated to sufficiently small reaction units, so that each reaction unit has only a single template molecule. After PCR reaction amplification, the droplet reader reads the droplet and calculates the ratio of the copy number concentration of the internal and external genes to achieve accurate quantification Construction and application of detection method for transgenic soybean ZH10-6. Background technique [0002] The development of genetically modified crops is advancing by leaps and bounds. Many countries and regions have implemented labeling management measures and set thresholds. For example, the Europea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q2600/13C12Q2600/166C12Q2531/113C12Q2545/101C12Q2561/101C12Q2563/159
Inventor 王一衡赵新刘双尉万聪兰青阔王永
Owner 天津市农业科学院