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

A technology for processing products and kits, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as cotton not being identified, and achieve the effect of protecting the right to know and eliminating test differences.

Inactive Publication Date: 2007-02-14
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

[0125] Embodiment 1, the preparation of trans CP4-EPSPS gene anti-glyphosate cottonseed standard reference sample

[0126] In the present invention, DNA is extracted from pure transgenic CP4-EPSPS gene-resistant glyphosate cottonseed and non-transgenic cottonseed by CTAB method, and both are diluted to 50ng / ul, and the two kinds of DNA are mixed according to the volume ratio to prepare transgenic glyphosate-resistant cottonseed Cottonseed DNA content accounted for 5%, 2%, 1%, 0.5%, 0.1%, 0.05% of the DNA solution, as a reference sample for quantitative detection.

Embodiment 2

[0127] Embodiment 2, the test kit for detecting trans CP4-EPSPS gene cottonseed and its processed products

[0128] The kit contains:

[0129] (1) A pair of specific primers for amplifying the SAD1 gene:

[0130] Upstream primer 5'CCA CGA GAC AGC CTA TAC CAA AA 3'

[0131] Downstream primer 5'CTT CTT CAT CAT GTC AGC AAA TGC 3'

[0132] 100μl each for 100uM;

[0133] (2) 100ng of probes that specifically bind to the amplification product of the SAD1 gene:

[0134] 5'CGT TGA AAA GCT GTT TGA GAT TGA TCC TGA TG 3';

[0135] (3) a specific primer pair for amplifying the CP4-EPSPS gene;

[0136] Upstream primer 5'CTG CTT TCC CAT TGG TTG CT 3'

[0137] Downstream primer 5'ACC AGT ACG GGT TGG GTT CA 3'

[0138] 100μl each for 100uM;

[0139] (4) 100ng of probes that specifically bind to the CP4-EPSPS gene amplification product:

[0140] 5'TTC CAG GTT CCG ACG TCA CCA TCC 3';

[0141] (5) Standard reference sample:

[0142] The CP4-EPSPS gene-transferred glyphosate-resistant ...

Embodiment 3

[0146] Embodiment 3, carry out qualitative detection with the kit of embodiment 2

[0147] Proceed as follows:

[0148] (1) Extract the DNA in the cottonseed to be tested with the magnetic bead method DNA extraction kit produced by Promega Company as a template;

[0149] (2) The test kit in Example 2 is detected, and the template DNA is added to the respective reaction systems for amplifying the SAD1 gene and the CP4-EPSPS gene, and incubated on a PCR amplification instrument to amplify the SAD1 gene and the CP4-EPSPS gene respectively;

[0150] Qualitative PCR reaction system (25μl reaction system) for amplifying SAD1 gene and CP4-EPSPS gene, the components of which are: 10×PCR reaction buffer 2.5μl, 10×25mMMgCl 2 2.5 μl, 10 mM dNTP 2.5 μl, Taq DNA polymerase 0.2 μl, 5 μM upstream primer 0.5 μl, 5 μM downstream primer 0.5 μl, sterilized ultrapure water 15.3 μl, added template DNA 1 μl (about 30 ng DNA).

[0151] Qualitative PCR cycle parameters are:

[0152] Pre-denaturat...

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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 invention relates to product detection technology, more specifically, the invention relates to a detection kit and detection method for qualitative and quantitative detection of 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 improved the yield and quality of agricultural products, and to a certain extent alleviated population growth, lack of natural resources, The reduction of arable land area and the impact of unpredictable natural disasters, etc. However, the safety of genetically modified crops has aroused great concern. [0003] Many countries in the world, such as the European Union, Japan, Australia, Singapore and other countries, have adopted legislation or other forms to strictly ma...

Claims

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Application Information

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