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Marker set and composition for comprehensively screening genetically modified ingredients and application

A technology of markers and compositions, applied in the biological field, can solve problems such as the lack of comprehensive evaluation of transgenic components or transgenic strain methods

Inactive Publication Date: 2019-07-12
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently, GMO testing is mostly for certain genes in certain breeds, and there is no method to comprehensively evaluate all GMO ingredients or GMO lines

Method used

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  • Marker set and composition for comprehensively screening genetically modified ingredients and application
  • Marker set and composition for comprehensively screening genetically modified ingredients and application
  • Marker set and composition for comprehensively screening genetically modified ingredients and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Design and screening of embodiment 1, primers and probes

[0093] According to a large amount of market research data, first determine all the commercially grown transgenic strains on the market, and then determine the following seven screening elements as screening elements covering the strains except maize DAS40278, soybean DP305423 and soybean CV127: PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9 and PAT; finally, query and determine the line-specific sequences of three transgenic lines: maize DAS40278, soybean DP305423 and soybean CV127.

[0094] Based on the above-mentioned 7 kinds of screening element sequences and the specific sequences of the above-mentioned three kinds of transgenic lines (10 kinds of sequences in total), the insertion sequence information of commercialized transgenic lines was collected, and based on this, each group (each screening element or Strain-specific sequences are grouped into one group, a total of 10 groups) and multiple pairs of prim...

Embodiment 2

[0117] Embodiment 2, the method for using digital PCR to detect genetically modified components

[0118] In this embodiment, it is judged whether the biological sample contains genetically modified components according to the result of digital PCR amplification. The method uses the 10 primers and probe combinations screened in Example 1 to carry out.

[0119] With the genome of the sample to be tested as a template, each sample to be tested is set in three parallels, and the combination of primers and probes (SEQ ID No.1-30) obtained by screening in Example 1 is used for digital PCR amplification, and negative Control and blank control, wherein negative control: use negative genome (ie, genomic DNA of non-transgenic sample) as template, and blank control: use water as template.

[0120] The specific steps of digital PCR amplification are as follows:

[0121] (1) Prime step: Remove the blue protective film from the digital PCR chip, inject Control Line Fluid into the holes on ...

Embodiment 3

[0135] Embodiment 3, sensitivity detection

[0136] Sensitivity verification was performed according to the method established in Example 2 for detecting transgenic components using digital PCR. The sensitivity of the detection system is defined as the lowest copy number that the detection system can perform stable detection. In this example, seven screening elements PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9, PAT and corn DAS40278, soybean DP305423, soybean CV127 were used The transgenic samples of the strain-specific sequences of the above-mentioned strains were artificially diluted to prepare homogeneous samples containing transgenic components of different mass percentages, and then digital PCR amplification was carried out according to the method of Example 2, and the specific steps were as follows:

[0137] (1) Test samples: select transgenic samples: corn MON87427 (as a sample for detection and screening element "PCAMV35S"), corn 59122 (as a sample for detection and ...

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Abstract

The invention discloses a marker set and composition for comprehensively screening genetically modified ingredients and application. The marker set comprises seven screening elements including PCAMV35S, TCAMV35S, TNOS, PRbcS4, TPINII, TE9 and PAT; the marker set further comprises strain-specific sequences of corn DAS40278, a soybean DP305423 and a soybean CV127. The composition is a reagent for detecting the marker set, preferably primers and probes shown in SEQ ID No.1-33. The marker set and composition can cover 66 commercial genetically modified strains on the market, have the advantages ofgood repeatability, high throughput, sensitivity, accuracy and rapidity, and have good application prospects in the aspect of detecting the genetically modified components.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a marker group, composition and application for comprehensively screening transgenic components. Background technique [0002] Genetically modified crops have been commercialized for more than 20 years. In 2016, the planted area reached 185.1 million hectares, and more than 26 countries have planted genetically modified crops. With the large-scale cultivation and consumption of genetically modified crops around the world, more and more countries and regions have begun to implement genetically modified product labeling management systems. In view of the inevitable mixing of genetically modified crops in the process of planting and harvesting, production, processing, transportation and consumption, more than 60 countries and regions have set thresholds for the labeling of genetically modified products. Relevant departments in our country are also studying to introduce it into the GMO ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/13C12Q2531/113
Inventor 付伟王晨光朱鹏宇朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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