Detection method and a kit of transgenic soybean MON89788 transformation event

A technology of MON89788 and genetically modified soybean, which is applied in the field of detecting whether there is a transformation event of genetically modified soybean MON89788 in biological samples, which can solve the problems of increasing the complexity of the test and mutual contamination of the experimental results, and achieves the reduction of detection cost, mutual pollution and easy degradation. Effect

Inactive Publication Date: 2013-11-27
海康生物科技(北京)有限公司 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above detection methods need to rely on expensive instruments, and because the amplification product is DNA, in order to prevent the mutual contamination of the experimental results caused by DNA, when designing the laboratory site, it is often necessary to combine sample extraction, sample amplification and sample detection. Distributed to different rooms and requires close and careful handling, which increases the complexity of the experiment

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  • Detection method and a kit of transgenic soybean MON89788 transformation event
  • Detection method and a kit of transgenic soybean MON89788 transformation event
  • Detection method and a kit of transgenic soybean MON89788 transformation event

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Construction of MON89788 plasmid standard molecule and design of detection primers and probes

[0034] Main reagents:

[0035]

[0036] A. Obtaining the target gene:

[0037] Genomic DNA extraction: Use the transgenic plant and deep-processed product extraction kit to extract the soybean genome to be tested. By designing specific primer pairs of SEQ ID No.9 and SEQ ID No.10 for the target gene fragment, optimizing the system and amplification program, the genome of soybean line MON89788 is amplified by PCR. (Amplification conditions: 95°C, 10min; 94°C for 30s, 58°C for 30s, 72°C for 30s (35 cycles); 72°C for 7min; 6°C for 1min).

[0038] PCR amplification, 25μl PCR reaction system is as follows: Add the following components in sequence in a 200μl small centrifuge tube

[0039]

[0040]

[0041] Add 2μl loading buffer to the PCR product, and electrophoresis.

[0042] After the amplification is completed, use an agarose gel recovery kit to recover and puri...

Embodiment 2

[0121] According to the MON89788 soybean genome NASBA method established in Example 1, by measuring 20 negative samples, negative soybeans (50ng / ul), other reagents, amplification conditions and amplification programs are unchanged, and the criticality of NASBA detection of transgenic soybean MON89788 is obtained. The critical value is 0.32 (Mean (negative sample)+10SD).

[0122]

[0123]

[0124] Example 3 Determination of MON89788 Genome Specificity and Sensitivity by NASBA Technology

Embodiment 3

[0125] 1. Determination of the specificity of MON89788 soybean genome by NASBA technology

[0126] In order to test the specificity of the MON89788 soybean NASBA detection method established in the research, the main transgenic crops that have been allowed to be commercialized are selected. These genetically modified samples basically include all transformation events of transgenic soybeans, corn, rapeseed, and cotton approved to enter the Chinese market as processing raw materials, and are used to verify that this detection method is only for a specific genetically modified line of MON89788. Constructed MON89788 positive plasmid, MON89788 positive genome, non-transgenic negative control, transgenic soybean mixed samples containing MON89788 (MON89788, GTS40-3-2, A5547-127, A2704-12), transgenic soybean mixed samples without MON89788 ( GTS40-3-2, A5547-127, A2704-12), transgenic corn mixture (MON810, MON863, Bt11, Bt176, NK603, GA21, TC1507, T25), genetically modified cotton mi...

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Abstract

The invention discloses a detection method and a kit of transgenic soybean MON89788 transformation event. The method comprises the following steps: performing high temperature degeneration on a sample DNA (deoxyribonucleic acid), transcribing under RNA (ribonucleic acid) polymerase by using a primer for identifying MON89788 specificity region to generate RNA, performing NASBA (nucleic acid sequence based amplification) by using the above primer based on obtained RNA as a template; and finally detecting the amplification product. Without purchasing large instrument, the detection cost of the method disclosed by the invention is reduced, the detected product is easy to degrade, and the problem of cross contamination among samples is reduced.

Description

technical field [0001] The invention relates to the field of transgenic plant detection, in particular to a method and a kit for detecting whether there is a transgenic soybean MON89788 transformation event in a biological sample. Background technique [0002] The successful introduction of commercially interesting traits into plants by genetic manipulation in agriculture or industry is a complex process dependent on different factors. According to statistics from the International Agricultural Bioengineering Applied Technology Acquisition Authority (ISAAA), since the commercialization of genetically modified products, the planting area of ​​genetically modified crops has exceeded 1 billion hectares. China has become the sixth largest growth country of GM crops. With the management of genetically modified products in various countries and people's attention to the safety of genetically modified products, countries around the world have issued relevant laws and policies to r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 于常海杨滨张明李飞武刘乐庭
Owner 海康生物科技(北京)有限公司
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