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Kit for testing transgene cottonseed and products produced, and testing method

A technology for processing products and kits, which is applied in the direction of biochemical equipment and methods, microbiological measurement/inspection, etc., can solve the problem that cotton has not been identified, and achieve the effect of protecting the right to know and eliminating test differences

Inactive Publication Date: 2005-11-16
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the internal standard genes of soybean, corn and rice have been determined, but the internal standard genes of cotton have not been determined
At present, there is no detection method and detection kit for detecting the content of genetically modified ingredients in genetically modified cottonseed and its processed products

Method used

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  • Kit for testing transgene cottonseed and products produced, and testing method
  • Kit for testing transgene cottonseed and products produced, and testing method
  • Kit for testing transgene cottonseed and products produced, and testing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1. Preparation of glyphosate-resistant cottonseed standard reference samples with CP4-EPSPS gene

[0125] In the present invention, the pure transgenic glyphosate-resistant cottonseed and non-transgenic cottonseed of the CP4-EPSPS gene are respectively extracted with DNA by the CTAB method, and both are diluted to 50ng / ul, and the two DNAs are mixed according to the volume ratio to form the transgenic glyphosate-resistant A DNA solution with cottonseed DNA content of 5%, 2%, 1%, 0.5%, 0.1%, and 0.05%, respectively, is used as a reference sample for quantitative detection.

Embodiment 2

[0126] Example 2. A kit for detecting transgenic cottonseed with CP4-EPSPS gene and its processed products

[0127] The kit contains:

[0128] (1) Specific primer pair for amplifying SAD1 gene:

[0129] Upstream primer 5’CCA CGA GAC AGC CTA TAC CAA AA 3’

[0130] Downstream primer 5’CTT CTT CAT CAT GTC AGC AAA TGC 3’

[0131] 100uM each 100μl;

[0132] (2) 100ng probe specifically binding to SAD1 gene amplification product:

[0133] 5’CGT TGA AAA GCT GTT TGA GAT TGA TCC TGA TG 3’;

[0134] (3) A pair of specific primers for amplifying the CP4-EPSPS gene;

[0135] Upstream primer 5’CTG CTT TCC CAT TGG TTG CT 3’

[0136] Downstream primer 5’ACC AGT ACG GGT TGG GTT CA 3’

[0137] 100uM each 100μl;

[0138] (4) 100ng of probe specifically binding to CP4-EPSPS gene amplification product:

[0139] 5’TTC CAG GTT CCGACG TCA CCATCC 3’;

[0140] (5) Standard reference sample:

[0141] The standard reference sample of glyphosate-resistant cottonseed transgenic CP4-EPSPS prepared in Example 1...

Embodiment 3

[0145] Example 3. Qualitative detection with the kit of Example 2

[0146] Proceed as follows:

[0147] (1) Use the magnetic bead DNA extraction kit produced by Promega to extract the DNA from the cottonseed to be tested as a template;

[0148] (2) For the detection in the kit of Example 2, the template DNA was added to the respective reaction systems for amplifying the SAD1 gene and CP4-EPSPS gene, and incubated on a PCR amplification machine to amplify the SAD1 gene and CP4-EPSPS gene respectively;

[0149] A qualitative PCR reaction system (25μ1 reaction system) for amplifying the SAD1 gene and CP4-EPSPS gene. The components are: 10×PCR reaction buffer 2.5μl, 10×25mMMgCl 2 2.5μl, 10mM dNTP 2.5μl, Taq DNA polymerase 0.2μl, 5μM upstream primer 0.5μl, 5μM downstream primer 0.5μl, sterilized ultrapure water 15.3μl, added template DNA 1μl (about 30ng DNA).

[0150] The qualitative PCR cycle parameters are:

[0151] Pre-denaturation at 95°C for 3min; denaturation at 94°C for 20s; ann...

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Abstract

A reagent kit for detecting the transgenic cotton seed and its products and its qualitative and quantitative test method aer disclosed. Said quantitative test method includes extracting the DNA as temple, PCR amplifications respectively by use of SADI specific primer pair and exogenous gene specific primer pair, respective hybridizations between amplified products and probe, measuring the signals generated by said hybridization and analyzing the data to obtain the content of transgenic component.

Description

Technical field [0001] The present invention relates to product detection technology. More specifically, the present invention relates to a detection kit and a detection method for qualitatively and quantitatively detecting genetically modified cottonseed and its processed products. Background technique [0002] In recent years, with the development of biotechnology, genetically modified crops have been rapidly commercialized and entered the international market through trade. The development of biotechnology has significantly increased the output and quality of agricultural products, and to a certain extent alleviated the impact of population growth, lack of natural resources, reduction of arable land area, and unexpected natural disasters. However, the safety of genetically modified crops has aroused great attention. [0003] Many countries in the world, such as the European Union, Japan, Australia, Singapore and other countries, have adopted legislation or other forms to stric...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 许文涛黄昆仑罗云波
Owner CHINA AGRI UNIV
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