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Positive plasmid molecule pBI121-Screening and application thereof

A technology of pbi121-screening and positive plasmids, which is applied in the direction of introducing foreign genetic material by using vectors, measurement/inspection of microorganisms, recombinant DNA technology, etc. It can solve the problem of inability to accurately reflect whether the sensitivity of detection parameters is consistent and meets standard requirements, and the difficulty of component detection , DNA extraction efficiency gap and other issues

Inactive Publication Date: 2015-05-27
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. Some special transgenic elements did not appear in legal transformation events, so there is no corresponding standard substance. For example, the hygromycin resistance gene HPT is commonly used in laboratory research, but it is completely absent in commercial varieties, resulting in The detection of this component is difficult;
[0006] 2. General GMO detection standard materials do not consider the presence or absence of each screening element and the copy number ratio. When multiple standard materials are used for GMO screening, the copy number of some elements may be several times that of other elements. , when used as a positive control, it may not accurately reflect whether the sensitivity of each detection parameter is consistent and meets the standard requirements;
[0007] 3. Different crops, or even different varieties of the same crop, may have a large gap in DNA extraction efficiency. In the development of EU standard materials for genetically modified products, it has been found that there is a difference of more than 30% in DNA extraction efficiency between genetically modified and non-transgenic raw materials
[0010] After searching the existing patents and other literatures, we have not found any reports on the development and application of universal positive plasmid molecules for screening and detection of genetically modified crops

Method used

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  • Positive plasmid molecule pBI121-Screening and application thereof
  • Positive plasmid molecule pBI121-Screening and application thereof
  • Positive plasmid molecule pBI121-Screening and application thereof

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Embodiment Construction

[0098] Below in conjunction with accompanying drawing and embodiment the present invention is described in detail:

[0099] 1. Construction of positive plasmid molecule pBI121-Screening

[0100] 1.1. Genomic DNA extraction from transgenic materials

[0101] First preheat 20% SDS to 65°C, take 15ml SDS extraction buffer (0.1TrisHCl, 0.05M EDTA, 1M NaCl pH8.0) into a 50ml centrifuge tube, then add 2.5μl β-mercaptoethanol, and mix well; Grind about 3g of leaves with nitrogen, transfer the powder to 50ml of a 50ml centrifuge tube containing extraction buffer, shake and mix on a shaker, add 2ml of preheated 20% SDS, mix well, and bathe at 65°C for at least 30 minutes, during which Shake the test tube gently; after the water bath, quickly place the centrifuge tube on ice, add 3ml 3M KAc, mix well, and place on ice for 30 minutes; centrifuge at 5000g at 4°C for 5min; transfer the supernatant to a new 50ml centrifuge tube , add 2 / 3 volume of isopropanol, mix well, place at -20°C for...

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Abstract

The invention discloses a positive plasmid molecule pBI121-Screening and application thereof and relates to the technical field of safety supervision and screening detection of transgenic crops in the transgenic safety field. The base sequence of the pBI121-Screening is as shown in SEQ NO.1. serving as both a positive contrast and a quality control sample, pBI121-Screening disclosed by the invention can be respectively used in general PCR and real-time fluorescence PCR screening detection of transgenic components of six transgenic crop samples, namely paddy rice, rapes, soybeans, corns, cotton and wheat. The pBI121-Screening disclosed by the invention can be used for solving the technical problem that the lack of a positive contrast or a standard sample in the screening detection of the transgenic crops, and prevents the problem that a plurality of positive contrasts are arranged for a plurality of detection targets in the screening detection, thus reducing the labor cost and economic cost for preparing the plurality of positive contrasts.

Description

technical field [0001] The invention relates to safety supervision and screening detection technology of transgenic crops in the field of transgenic safety, in particular to a positive plasmid molecule pBI121-Screening and its application. Background technique [0002] Over the past 20 years, the global R&D and commercialization of GMOs have developed rapidly. In 2013, 27 crops involving 336 transformants were approved for commercial production in the world, and the planting area exceeded 1.75 billion hectares, which produced huge economic benefits (James, Briefs No.46 http: / / www.isaaa.org / purchasepublications / itemdescription.asp? ItemType e=BRIEFS&Control=IB046-2013 , 2013). At the same time, the safety of genetically modified organisms has always attracted the attention of all walks of life. For this reason, more than 50 countries and regions, including China, the European Union, the United States, Japan and South Korea, have promulgated and implemented laws and regula...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/63
CPCC12Q1/6895C12Q2600/166
Inventor 吴刚武玉花李俊王宇蕾李晓飞李允静
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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