Visual quantitative screening new method for dual genes of transgenic crops

A screening and genetic technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of lack of official positive declaration, lack of scientificity, etc., and achieve the effect of simple and efficient screening and analysis

Inactive Publication Date: 2018-07-06
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above-mentioned controversies have been proved to lack the scientificity of experimental design, statistics or conclusions, or the lack of official positive statements, the doubts about the food and environmental safety of genetically modified organisms and their product ingredients have never ceased, and there are still growing concerns. trend

Method used

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  • Visual quantitative screening new method for dual genes of transgenic crops
  • Visual quantitative screening new method for dual genes of transgenic crops
  • Visual quantitative screening new method for dual genes of transgenic crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Embodiment 1, the establishment of a double colorimetric sensing method for screening the 35s promoter of cauliflower mosaic virus (CaMV) and the terminator of nopaline synthase gene (Nos) by ultra-fast PCR

[0081] (1) Experimental materials

[0082] The nucleotide sequences of the primers designed in this example are shown in Table 1 and the sequence listing.

[0083] Table 1

[0084]

[0085] In Table 1, the upstream primer 35s-F is obtained by carrying out biotin (Biotin) labeling at the 5' end of the nucleotide sequence shown in SEQ ID No. 1 in the sequence listing; the downstream primer 35s-R is obtained by marking the The 3' end of the nucleotide sequence shown in SEQ ID No.: 2 in the sequence listing is obtained after Cy5 labeling; the upstream primer Nos-F is obtained by the nucleotide sequence shown in SEQ ID No.: 3 in the sequence listing The 5' end was labeled with biotin (Biotin); the upstream primer Nos-R was labeled with Digoxin (Digoxin) at the 3' e...

Embodiment 2

[0110] Embodiment 2, sensitivity experiment

[0111] The transgenic crop CBH-351-corn seeds were ground into powder using a ball mill, and the genome was extracted using the plant genome extraction kit of Quanshijin Company, and the amplification reaction was carried out with reference to the double ultra-fast PCR reaction described in step (3) of Example 1 above, wherein The genomes that have been serially diluted are used as templates, and the amount of the templates, that is, the contents of the transgenic components after serial dilution are: (A) 100%, (B) 50%, (C) 5%, (D) 0.5%, ( E) 0.05%, (F) 0%, the percentages are mass percentages.

[0112] After the amplification reaction is completed, mix 10 μL of the dual ultra-fast PCR reaction system with 60 μL of detection buffer (4×SSC, 2% BSA by mass, 0.05% by mass Tween-20, pH 7.0) and mix evenly. Immerse the colloidal gold immunochromatography test paper prepared above, react at room temperature for 5 minutes, and observe th...

Embodiment 3

[0114] Embodiment 3, specificity experiment

[0115]The transgenic crops 59122-corn seeds, GA21-corn seeds, MON809-corn seeds, 32138-corn seeds, 3272-corn seeds, CBH-351-corn seeds, MON87411-corn seeds, 33121-corn seeds are ground into powder using a ball mill, Genomes were extracted using Quanshijin Plant Genome Extraction Kit, and the extracted genomes were used as templates (add 2 uL to each template), and the double ultra-fast PCR reactions were carried out with reference to the double ultra-fast PCR reaction described in step (3) of Example 1 above. .

[0116] After the amplification reaction is completed, mix 10 μL of the dual ultra-fast PCR reaction system with 60 μL of detection buffer (4×SSC, 2% BSA by mass, 0.05% by mass Tween-20, pH 7.0) and mix evenly. Immerse the colloidal gold immunochromatography test paper prepared above, react at room temperature for 5 minutes, and observe the results.

[0117] Experimental results such as Figure 4 and shown in Table 4. T...

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Abstract

According to a screening method based on superfast PCR (Polymerase Chain Reaction) and GICA (Gold Immunochromatographic Assay) test paper, and a biosensor, established by the invention, the overall screening time of transgenic crops is controlled in about 10 minutes; compared with a traditional method, the screening method is quicker and more flexible and has the advantages of strong specificity,high sensitivity, reliable detection results and the like, analysis and detection steps can be simplified, the analysis time can be shortened, more importantly, online in-time detection becomes possible, carrying and field operation are convenient, and the screening method has a very good application prospect in the field of rapid screening of the transgenic crops.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a new visualization method for quantitatively screening double genes of transgenic crops. Background technique [0002] Since the first commercial planting of transgenic tomato in the United States in 1994, transgenic technology has been rapidly applied and developed in the field of agricultural production. According to statistics from the International Agricultural Biotechnology Application Service Organization, in 2014 the number of countries planting genetically modified crops reached 28, and the global planting area of ​​genetically modified crops increased from 1.7 million hectares in 1996 to 181 million hectares in 2014. 100 times. With the rapid development of genetically modified crops, the controversy about the edible and environmental safety of genetically modified organisms and their product ingredients is also increasing day by day, and corre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6804C12Q1/6895
CPCC12Q1/6804C12Q1/6895C12Q2600/16C12Q2531/113C12Q2563/131C12Q2565/625C12Q2537/143
Inventor 许文涛黄昆仑罗云波田晶晶杜再慧
Owner CHINA AGRI UNIV
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