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Primers, probes and method for real-time PCR accurate detection of transgenic plants containing gat-tpinii structure

A technology of gat-tpinii-f1 and gat-tpinii-p2, applied in the field of quantitative analysis, can solve the problems of false positives and low accuracy, achieve high accuracy, high accuracy, and avoid false positives

Active Publication Date: 2019-01-04
四川省农业科学院分析测试中心
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Problems solved by technology

As we all know, the dye method does not involve specific probes, and the presence of any double-stranded nucleic acid fragments will generate fluorescent signals due to non-specific fuel intercalation. Therefore, non-specific amplification or the generation of primer-dimers during the PCR reaction may be blocked. As a positive signal, false positives occur, and the accuracy is low

Method used

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  • Primers, probes and method for real-time PCR accurate detection of transgenic plants containing gat-tpinii structure
  • Primers, probes and method for real-time PCR accurate detection of transgenic plants containing gat-tpinii structure
  • Primers, probes and method for real-time PCR accurate detection of transgenic plants containing gat-tpinii structure

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Embodiment

[0034] The real-time PCR accurate detection method for transgenic plants containing the gat-tpinⅡ structure, when detecting the junction of the gat gene and the pinⅡ terminator, the steps are as follows:

[0035] (a1) Synthesize the following primers and fluorescent probes used in conjunction with the primers,

[0036] Upstream primer sequence, gat-tpinⅡ-F1: 5'-AGTTRGGMTTCAGYGAGCARGGAGA-3';

[0037] Downstream primer sequence, gat-tpinⅡ-R: 5'-CCAATCCAGAAGATGGACAAGTC-3';

[0038] fluorescent probe sequence,

[0039] gat-tpin II-P2: 5'-FAM-CCKCCAGTWGGACCTCACATCCTGATGTAT-TAMRA-3'.

[0040] The synthesis concentrations of the primers and fluorescent probes in this example were both 10 μmol / l.

[0041] The nucleotide sequences of the above primers and fluorescent probes are designed based on the specific site where the gat gene is connected to the pinⅡ terminator in the construction structure; through this design, all transgenic plants and products containing the gat-tpinⅡ struc...

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Abstract

The invention belongs to the technical field of biology, relates to a quantitative analysis method of genes, and specifically relates to a method for real-time precise PCR (polymerase chain reaction) detection of transgenic plants containing a gat-tpin II structure. The quantitative PCR detection is carried out by a PCR reaction system prepared from a designed specific upstream primer sequence, a downstream primer sequence, a fluorescent probe sequence, a DNA diluent, Taqman Master mix (2x) and water. A Taqman quantitative PCR detection technology with high amplification efficiency and high accuracy is mainly established, and is suitable for domestic agricultural transgenic organism and product supervision and inspection, the inspection of transgenic organisms and products at entry and exit ports, and the detection of biological components containing the gat-tpin II structure of raw materials imported within enterprises.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a quantitative analysis method of genes. Background technique [0002] With the development of transgenic technology, the global planting area of ​​transgenic plants has accumulated more than 2 billion hectares. However, it has not entered commercial development in many countries and regions, and is still in a restrictive development stage. Many countries in the world implement limited labeling and importation of genetically modified products, but there is no specific labeling threshold for genetically modified products in my country. As the types and quantities of genetically modified plants increase, the requirements for the development of genetically modified ingredient content detection and risk assessment technologies continue to increase and improve, and the importance of screening methods is also becoming more and more prominent. For example, detection technologies for the spec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2561/101
Inventor 张富丽常丽娟尹全宋君王东郭灵安雷绍荣
Owner 四川省农业科学院分析测试中心
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