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Transgenic maize T4-1-1 strain specific detection method and reagent kit

A genetically modified corn, specific technology, applied in the direction of biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc.

Active Publication Date: 2015-12-23
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

There is currently no line-specific PCR assay for transgenic maize T4-1-1

Method used

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  • Transgenic maize T4-1-1 strain specific detection method and reagent kit
  • Transgenic maize T4-1-1 strain specific detection method and reagent kit
  • Transgenic maize T4-1-1 strain specific detection method and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Screening of strain-specific qualitative detection primers for transgenic maize T4-1-1

[0033] 1. Design of primers

[0034] Design 4 pairs of transformant-specific primers for the target sequence according to the junction region sequence (SEQ ID No.9) inserted into the gene vector and its 3' side sequence according to T4-1-1, and the primer sequences are as follows:

[0035] Primer 1: The size of the amplified product fragment is 293bp

[0036] 1-F: TCGTGAGAGTTTAGCGATTGG (SEQ ID No. 1)

[0037] 1-R: ATGACGTTATTTATGAGATGGGTTT (SEQ ID No. 2)

[0038] Primer 2: The size of the amplified product fragment is 176bp

[0039] 2-F: GAATCTTAGCTGTCCATGTTGGG (SEQ ID No. 3)

[0040] 2-R: GGATAAATTATCGCGCGCG (SEQ ID No. 4)

[0041] Primer 3: The size of the amplified product fragment is 270bp

[0042] 3-F: AATGGAATCTTAGCTGTCCA (SEQ ID No. 5)

[0043] 3-R: GACGTTATTTATGAGATGGGTTTTTT (SEQ ID No. 6)

[0044] Primer 4: The size of the amplified product fragment is 122...

Embodiment 2

[0049] Example 2 T4-1-1 strain-specific qualitative PCR detection method established based on the flanking sequence of the exogenous insert fragment of the transgenic maize T4-1-1 strain

[0050] 1. Extraction and detection of plant genomic DNA

[0051] 1 Plant DNA Extraction

[0052] (1) 100mg of sample is ground into powder and transferred to a 2mL centrifuge tube;

[0053] (2) 1mL of CTAB extraction buffer preheated to 65°C, mix thoroughly, suspend the sample, and mix gently, put it in a water bath at 65°C for 40 minutes, and invert and mix several times during the period;

[0054] (3) Centrifuge at 12000×g for 15 minutes. Transfer the supernatant to a new centrifuge tube, add an equal volume of phenol, chloroform-isoamyl alcohol (24:1), and mix well. Repeat this operation once;

[0055] (4) Centrifuge at 12000×g for 10 min, take the supernatant, add 2 / 3 volume of isopropanol, and 1 / 10 volume of sodium acetate. Store at -20°C for 2 hours or longer;

[0056] (5) Centri...

Embodiment 3T4-1-1

[0073] Specificity Analysis of Example 3 T4-1-1 Transgenic Maize Line-Specific Qualitative PCR Detection Method

[0074] 1. Experimental materials

[0075] Sample 1: Non-transgenic corn Jidan 180, Jidong 26, Shenyu 21, and Yuqing 281 were mixed in equal amounts to make a sample.

[0076] Sample 2: Negative for rice.

[0077] Sample 3: Negative for soybeans.

[0078] Sample 4: Negative for cotton.

[0079] Sample 5: Rapeseed negative.

[0080] Sample 6: transgenic corn GA21, NK603, T25, TC1507, Bt11, Bt176, MON863, MON89034, MIR604, mixed to make a sample, each content 5%.

[0081] Sample 7: Transgenic rice Kefeng No. 6, Kemo rice, M12, TT51 were mixed to make a sample, each with 5% content.

[0082] Sample 8: Transgenic soybeans 356043, 305423, CV127, MON89788, A2704-12, GTS40-3-2 were mixed to make one sample, each with 5% content.

[0083] Sample 9: Transgenic cotton MON1445, MON88913, LLCOTTON25, MON15985 were mixed to make one sample, each with 5% content.

[0084] ...

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Abstract

The invention designs four pairs of PCR detection primers used for detecting the strain specificity of transgenic maize T4-1-1 and further provides a reagent kit containing the primers. Experiments indicate that by means of the detection primers, the PCR qualitative detection conducted on the strain specificity of the transgenic maize T4-1-1 is great in specificity and high in sensitivity.

Description

Technical field: [0001] The invention relates to a line-specific detection of transgenic crops, in particular to a line-specific PCR detection method and kit for transgenic corn T4-1-1. Background technique: [0002] Qualitative detection of components in transgenic plants and their processed products can be divided into screening detection methods, gene-specific detection methods, and strain-specific detection methods according to the specificity of detection. [0003] Strain-specific detection is realized by detecting the sequence of the junction region between the insertion vector and the plant genome, since each transgenic plant line has a specific junction region sequence between the foreign insertion vector and the plant genome. Strain specificity has higher specificity and accuracy, and is most suitable for the detection of genetically modified products. [0004] According to the national standard "Detection of Genetically Modified Plants and Their Products", strain-...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 周正富张维余桂容徐利远林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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