Primer for genetically modified alfalfa J163 strain specificity qualitative PCR (Polymerase Chain Reaction) detection and genetically modified alfalfa J163 strain specificity qualitative PCR detection method

A transgenic alfalfa, J163 technology, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problem of inability to identify transgenic products, lack of strain-specific detection methods for transgenic alfalfa, and inability to identify, etc. problems, achieving stable qualitative detection, good specificity, and simple operation

Inactive Publication Date: 2015-02-25
中华人民共和国黄埔出入境检验检疫局
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a transgenic alfalfa in view of the lack of strain-specific detection methods for the existing transgenic alfalfa, the inability to accurately identify the strain of the transgenic alfalfa, and the inability to identify the transgenic product. The primers for J163 strain-specific qualitative PCR detection have high specificity, sensitivity and good stability. Based on the primers, a specific qualitative PCR detection method for transgenic alfalfa J163 strains can be successfully established to realize the detection of transgenic alfalfa J163 Qualitative testing of strains

Method used

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  • Primer for genetically modified alfalfa J163 strain specificity qualitative PCR (Polymerase Chain Reaction) detection and genetically modified alfalfa J163 strain specificity qualitative PCR detection method
  • Primer for genetically modified alfalfa J163 strain specificity qualitative PCR (Polymerase Chain Reaction) detection and genetically modified alfalfa J163 strain specificity qualitative PCR detection method
  • Primer for genetically modified alfalfa J163 strain specificity qualitative PCR (Polymerase Chain Reaction) detection and genetically modified alfalfa J163 strain specificity qualitative PCR detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 : Establishment of J163 strain-specific qualitative PCR detection system

[0035] Design primers:

[0036] The present invention summarizes the contiguous region sequence between the exogenous insertion fragment P-eFMV at the 5' end of the transgenic alfalfa J163 strain and the alfalfa genomic DNA through creative analysis and a large number of experimental studies, combined with the analysis of sequence biological information, the design A pair of specific primers and probes are obtained. And determined the precise detection conditions, and established a qualitative PCR detection method for the transgenic alfalfa J163 strain with strong specificity, high sensitivity and good stability.

[0037]Specifically, a transgenic alfalfa J163 strain-specific qualitative PCR detection primers J163-1 and J163-2 are provided, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2:

[0038] SEQ ID NO.1: J163-1: 5'-GGACAAGGTCATCCAAACTGA-3',

[0039] SEQ ID NO....

Embodiment 2

[0047] Example 2: Specific test of J163 qualitative PCR

[0048] The present invention uses a variety of crops to conduct specificity experiments, and the following examples illustrate 12 crops including J163: J163, transgenic corn line MIR162, transgenic corn line 89034, genetically modified corn line NK63, genetically modified corn line BT11, and transgenic rice (cry IA(a / b)), GM soybean GM soybean GTS40-3-2, non-GM rapeseed meal, non-GM wheat, non-GM palm meal, non-GM alfalfa, non-GM rice (all from routine random sampling After storage, this does not limit the scope of the present invention) Genomic DNA is used as a template, and the transgenic alfalfa J163 strain-specific qualitative PCR method established in the present invention is used for amplification, and the specificity of the qualitative PCR method established in the present invention is tested. The experimental results are attached figure 2 As shown, electrophoresis analysis after PCR amplification was used to...

Embodiment 3

[0051] Example 3 : Sensitivity test of J163 qualitative PCR

[0052] The transgenic alfalfa J101 strain-specific qualitative PCR method established by the present invention is used for amplification, using 100ng / μL, 16ng / μL, 1.6ng / μL, 0.16ng / μL, 0.08ng / μL, 0.016ng / μL and 0.008ng / μL μL of the genomic DNA of the J163 strain was used as a template for detection, and the experimental results were as follows: image 3 shown. When the template amount is not less than 0.08ng, a specific 208bp fragment can be amplified. It shows that its sensitivity is 0.08ng of genomic DNA, which is equivalent to 50 copies of genomic DNA.

[0053] The reaction system for qualitative PCR detection is 25 μL: Takara Ex Taq 12.5 μL, primers J163-1 and J163-2 each 1 μL, DNA template 1 μL, ddH 2 O 10.5 μL.

[0054] The reaction program of qualitative PCR detection described in S2 is: 98°C for 10s, 61°C for 30s, 72°C for 30s, 30 cycles; the amplified products were analyzed by 1.5% agarose gel electrop...

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Abstract

The invention discloses a primer for genetically modified alfalfa J163 strain specificity qualitative PCR (Polymerase Chain Reaction) detection and a genetically modified alfalfa J163 strain specificity qualitative PCR detection method. According to the invention, a pair of specific primers are firstly designed and provided according to an adjacent area positioned between the 5'end exogenous insertion element P-eFMV of a genome DNA sequence and alfalfa genome DNA aiming at a genetically modified alfalfa J163 strain without an agriculture genetically modified organism safety certificate issued by the national department of agriculture, and a qualitative PCR detection system is successfully established. A result shows that the qualitative PCR detection method disclosed by the invention has good specificity and is suitable for fast and stable qualitative detection of the J163 strain, and detected lowest DNA concentration is 80 pg and is equivalent to genome DNA of 50 copies.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a primer and a detection method for qualitative PCR detection specific to a transgenic alfalfa J163 strain. Background technique [0002] The genetically modified alfalfa J163 strain is a herbicide-resistant genetically modified strain developed by Monsanto. In 2005, the United States and Canada approved its environmental release and application as feed. At present, my country has approved its import. Has not yet obtained my country's GMO safety certificate. Genetically modified alfalfa enters the food chain of animal breeding through feed, causing milk, meat and other products to become genetically modified food, which may have an impact on human health. According to the "Administrative Measures for the Labeling of Agricultural Genetically Modified Organisms", all genetically modified organisms sold in China must be labeled. In order to protect consumers' right to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/158
Inventor 刘二龙卢丽吕英姿蒋湘唐婕林惠娇樊武疆姚柏辉王定国苏彩珠郑高彬林学勤秦焯敏
Owner 中华人民共和国黄埔出入境检验检疫局
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