Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes

A technology of exogenous genes and genes, applied in the field of transgenic detection, can solve the problems of complex development process of matrix reference materials, difficulty in obtaining plant materials, and no commercial production, etc., so as to facilitate transgenic monitoring and safety supervision, and the construction is simple and fast , low-cost effect

Active Publication Date: 2021-04-13
HUAZHI RICE BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are hundreds of genetically modified strains that have passed safety evaluation or have been commercialized in various countries around the world, but many genetically modified strains have not been commercially produced in China
It is difficult to obtain plant materials of these strains. The current commercial GMO detection standards are mainly matrix reference materials. The development process of matrix reference materials is relatively complicated and the price is very expensive. Each gram of matrix reference material powder is about 100-200 euros
Therefore, the purchase of reference materials is a heavy burden for institutions that want to conduct genetically modified testing
In addition, the development of domestic standard products is relatively lagging behind. If you want to buy other standard products, you need to import them. Because of the genetic modification, the procedures are cumbersome and the cycle is long.

Method used

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  • Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes
  • Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes
  • Plasmid standard molecule capable of being used for detecting multiple crops and multiple exogenous genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044]Example 1 Construction of Plasmid Standard Molecules

[0045]1. Inquiry of exogenous genes and internal standard gene DNA sequences

[0046]Selecting the 39 foreign genes in transgenic breeding and 13 standard genes in 13 major crops and economic crops, and 39 foreign genes are shown in Table 1, and the standard gene is shown in Table 2, searching for these in GenBank. Gene's DNA sequence. Determination of detection target sequence SEQ ID NO: 4 ~ SEQ ID NO: 55 of each gene is determined based on the methods disclosed in China and EU genetically modified biological testing technology standards and have been verified.

[0047]Table 1 39 common foreign genes

[0048]

[0049]

[0050]

[0051]Table 2 13 standard genes

[0052] Internal standard gene name Gene source Sequence sequence ZSSIIB corn SEQ ID NO: 43 Tswaxy wheat SEQ ID NO: 44 STLAT52 tomato SEQ ID NO: 45 Sialb sesame SEQ ID NO: 46 Ossps Rice SEQ ID NO: 47 MSACC Alfalfa SEQ ID NO: 48 Lusad flax SEQ ID NO: 49 JRVIC peanut SEQ ID NO: 5...

Embodiment 2

[0057]Example 2 Primer information

[0058]For P-MTL, P-OCS, P-PEPC, P-RBCS, etc. Promoters and T-TML, corn T-GLB1, Soy T-7SUTR, T-PEPC, Potato T-Pinii, T-TR7, T-VSP The et alted sub-gene elements have also been developed to detect the primers for fluorescent dye method for these genes, see Table 3.

[0059]Table 3 Fluorescent dye method PCR detection primers

[0060]

Embodiment 3

[0061]Example 3 Application of standard plasmid molecules

[0062]Real-time fluorescent PCR amplification was performed by plasmid molecular plangutou-5 as a template molecule plangutou-5 using a real-time fluorescent PCR primer and Taqman probe in a standard gene in which the standard gene of the synthesis of 39 foreign gene elements and 13 crops were used. Amplification curvefigure 2 , 39 amplification curves of the original exogenous geneimage 3 .

[0063]Fromfigure 2 withimage 3 It can be found that each gene element produces a typical amplification curve, indicating that the plasmid molecule plangutou-5 contains real-time fluorescent PCR detecting the target. Plasmid molecules Plangutou-5 can be used as positive control and quality control samples for 13 transgenic crops real-time fluorescent PCR screening.

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Abstract

The invention discloses a plasmid standard molecule pLANGUTOU-5 capable of being used for detecting multiple crops and multiple exogenous genes and application thereof. The base sequence of the plasmid standard molecule is shown as SEQ ID NO: 1. The pLANGUTOU-5 is used as a positive control or a positive standard substance, 13 species and 39 exogenous genes can be detected, the problem that a transgenic positive material is difficult to obtain is solved, the exogenous gene detection range is greatly enlarged, the risk of missing detection is reduced, and the requirement for preparing multiple positive controls is also reduced. The plasmid constructed through the method can be applied to 13 main crops and economic crops, the variety of detected crops is greatly enlarged, and transgenic monitoring and safety supervision are facilitated. The plasmid molecule provided through the method is easy and fast to construct and low in cost, and the transgenic detection cost can be greatly reduced.

Description

Technical field[0001]The present invention belongs to the field of transgenic detection, and is specifically involved in a plasmid standard molecule which can be used to detect a variety of crops and a plurality of foreign genes.Background technique[0002]GM product safety has been attracted, and the molecular detection of transgenic crops and its products is increasingly important. At present, countries have different requirements for genetically modified products, and some national provisions must indicate the content of transgenic components, and the requirements of each country have different requirements for the identified transgenic content. In order to solve the problem of trade disputes, the qualitative and quantitative detection of transgenic products become critical (Dong Lianhua, etc.). The detection method of currently used transgenic crops is based on sample DNA detection methods and method based on sample proteins. Sample protein-based detection methods include Enzyme-L...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12Q1/6895C12Q1/686C12R1/19
CPCC12N15/70C12Q1/6895C12Q1/686C12Q2600/166C12Q2563/107C12Q2561/113Y02A40/146
Inventor 肖金华杨翅春朱丹彭佩郑秀婷唐顺学田冰川
Owner HUAZHI RICE BIO TECH CO LTD
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