Qualitative PCR (polymerase chain reaction) detection primers and detection method for specificity of transgenic alfalfa J101 strain

A technology for transgenic alfalfa and detection methods, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., and can solve the problems of inability to identify transgenic alfalfa, identification of transgenic alfalfa, and no strain specificity of transgenic alfalfa In order to achieve the effect of stable qualitative and quantitative 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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Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a transgenic alfalfa for the existing transgenic alfalfa without a strain-specific detection method, the inability to accurately identify the strain of the transgenic alfalfa, and the inability to identify the transgenic product. The primers for J101 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 J101 strains can be successfully established to realize the detection of transgenic alfalfa J101 Qualitative testing of strains

Method used

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  • Qualitative PCR (polymerase chain reaction) detection primers and detection method for specificity of transgenic alfalfa J101 strain
  • Qualitative PCR (polymerase chain reaction) detection primers and detection method for specificity of transgenic alfalfa J101 strain
  • Qualitative PCR (polymerase chain reaction) detection primers and detection method for specificity of transgenic alfalfa J101 strain

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 : Establishment of J101 strain-specific qualitative PCR detection system

[0034] Design primers:

[0035]The present invention summarizes the contiguous region sequence between the exogenous insertion fragment P-eFMV at the 5' end of the transgenic alfalfa J101 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 J101 strain with strong specificity, high sensitivity and good stability.

[0036] Specifically, a transgenic alfalfa J101 strain-specific qualitative PCR detection primers J101-1 and J101-2 are provided, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively:

[0037] SEQ ID NO. 1: J101-1: 5'-CGTATTCATGTCATGTGTTTTGTACTG-3'. ...

Embodiment 2

[0049] Example 2: Specific test of J101 qualitative PCR

[0050] In the present invention, various crops are used to carry out the specificity experiment, and seven kinds of crops are taken as examples below for illustration. The genetically modified corn line MIR162, the genetically modified corn line 89034, the genetically modified corn line NK63, the genetically modified corn line BT11, the genetically modified soybean GTS40-3-2, and the non-transgenic alfalfa (all come from routine random sampling and storage, and are not limited thereby) The scope of the present invention) and alfalfa J101 genomic DNA were used as templates, and the transgenic alfalfa J101 line-specific qualitative PCR method established in the present invention was used to amplify, and the specificity of the qualitative PCR method established in the present invention was tested. The experimental results are attached figure 2 As shown, the presence or absence of a 170bp band was observed by electropho...

Embodiment 3

[0053] Example 3 : Sensitivity test of J101 qualitative PCR

[0054] The transgenic alfalfa J101 strain-specific qualitative PCR method established by the present invention is used for amplification, and the J101 strains of 100ng / μL, 16ng / μL, 0.16ng / μL, 0.08ng / μL, 0.016ng / μL and 0.008ng / μL are used. Genomic DNA is used as a template for detection, and the experimental results are as follows: image 3 shown. When the template amount is not less than 0.08ng, a specific 201bp fragment can be amplified. It shows that its sensitivity is 0.08ng of genomic DNA, which is equivalent to 50 copies of genomic DNA.

[0055] The qualitative PCR reaction system is 25 μL: Takara Ex Taq12.5 μL, J101-1 / 2 respectively 0.5 μL, DNA template 1 μL, ddH 2 O 10.5 μL;

[0056] The qualitative PCR reaction program was: 98°C for 10s, 60°C for 30s, 72°C for 30s, 30 cycles. Amplified products were analyzed by 1.5% agarose gel electrophoresis.

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Abstract

The invention discloses qualitative PCR (polymerase chain reaction) detection primers and a detection method for specificity of a transgenic alfalfa J101 strain. A pair of specific primers is designed and provided for the first time and a qualitative PCR detection system is successfully established according to an adjacent area between a 5'-end exogenous insert fragment P-eFMV of a genome DNA sequence of the transgenic alfalfa strain J101 which is not issued with an agricultural transgene bio-safety certificate by the agriculture department of China and an alfalfa genome DNA. Results show that the qualitative PCR detection method disclosed by the invention has good specificity; the detected lowest DNA concentration is 80pg, which is equivalent to genome DNA of 50 copies; and the detection method is suitable for quick and stable qualitative detection of the J101 strain.

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 J101 strain. Background technique [0002] At present, there are 15 million dairy cows in my country, and the demand for high-quality forage is the greatest. 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 know, the genetically modified product labeling system is applied in more than 30 countries and regions. China currently implements a mandatory labeling system with no threshold. [0003] The identification of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 卢丽刘二龙吕英姿蒋湘唐婕樊武疆姚柏辉王定国林惠娇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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