Supercharge Your Innovation With Domain-Expert AI Agents!

Primer and probe combination for double nested fluorescent PCR detection of transgenic soybean DAS44406A and method and kit

A technology of transgenic soybeans and kits, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of low detection throughput and unstable reaction, and achieve high sensitivity, wide applicability, Good repeatability and specificity

Inactive Publication Date: 2017-01-04
中华人民共和国湛江出入境检验检疫局 +1
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the European Union's genetically modified standards have introduced a fluorescent PCR detection method for genetically modified soybean DAS44406, this method has quantitative capabilities, but its detection throughput is low due to the probe; Detection throughput, but the system contains many pairs of primers, and the reaction is unstable

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Primer and probe combination for double nested fluorescent PCR detection of transgenic soybean DAS44406A and method and kit
  • Primer and probe combination for double nested fluorescent PCR detection of transgenic soybean DAS44406A and method and kit
  • Primer and probe combination for double nested fluorescent PCR detection of transgenic soybean DAS44406A and method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] This example provides primer and probe combinations and their applications.

[0057] The screening method of primer and probe combination is: for transgenic soybean DAS44406 exogenous gene 5' insertion sequence (such as figure 1 Shown) and the soybean endogenous gene Lectin, designed different primers and probe combinations and carried out optimized screening, integrated its specificity, sensitivity, interaction of paired multiplex amplification, and different primer-probe combinations and fluorescent PCR amplification The adaptability of the kit, and finally screen out the following primer pairs and probe combinations with good specificity, good repeatability and high sensitivity for dual nested fluorescent PCR that can simultaneously detect the specific sequence of the transgenic soybean DAS44406 line and the soybean endogenous gene .

[0058] The nucleotide sequence of described primer and probe combination is as shown in table 1:

[0059] Table 1

[0060]

[0...

Embodiment 2

[0066] This embodiment provides a kit and application thereof, the kit comprising:

[0067] (1) SEQ ID NO: 1-SEQ ID NO: 10 (from Example 1);

[0068] (2) dNTPs, MgCl 2 , at least one of 10×PCR Buffer reaction buffer and DNA polymerase;

[0069] (3) Genomic DNA extraction reagents;

[0070] (4) Probe qPCR Mix;

[0071] (5) Positive control and negative control.

[0072] The above dNTPs, MgCl 2 , 10×PCR Buffer reaction buffer and DNA polymerase are all reagents in the kit with the product number RR060A from Treasure Bioengineering (Dalian) Co., Ltd.

[0073] The above-mentioned genomic DNA extraction reagents come from Tiangen Biochemical Technology (Beijing) Co., Ltd. and the reagents in the plant genomic DNA extraction kit with the article number DP305-02.

[0074] The above-mentioned Probe qPCR Mix comes from the reagents in the kit with the product number RR391S from Treasure Bioengineering (Dalian) Co., Ltd.

[0075] Experiments have proved that the above-mentioned g...

Embodiment 3

[0078] This example provides a method for detecting or assisting in the detection of transgenic soybean DAS44406, for example, judging whether the biological sample contains the line-specific sequence of transgenic soybean DAS44406 and / or the soybean endogenous gene Lectin according to the results of double-nested fluorescent PCR amplification. The method uses the primer and probe combination of Example 1 or the kit of Example 2.

[0079] Above-mentioned method comprises the steps:

[0080] (1) Extraction of genomic DNA

[0081] Genomic DNA of biological samples was extracted using the Plant Genomic DNA Extraction Kit with the article number DP305-02 from Tiangen Biochemical Technology (Beijing) Co., Ltd. After extraction, 1 μL was used to measure the concentration and purity of the extracted genomic DNA with a nucleic acid protein analyzer. TE calibrated all samples to 100ng / μL and stored at -20°C until use.

[0082] (2) Double nested fluorescent PCR amplification

[0083]...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a primer and probe combination for double nested fluorescent PCR detection of endogenous and exogenous genes of transgenic soybean DAS44406A. In the invention, double PCR, nested PCR and fluorescent quantitative PCR are combined, and the design of primer and probe is strictly controlled; the detection flow is improved, and a double nested fluorescent quantitative PCR detection system is established and can qualitatively and quantitatively detect the specific sequence of the transgenic soybean DAS44406A strain and the soybean endogenous gene Lectin; and the sensitivity of the method is improved by 1 order of magnitude in comparison with common fluorescent quantitative PCR. The method disclosed by the invention has the advantages of low template need, high sensitivity, high flux, strong specificity and the like, can be applied to quick quantitative detection of transgenosis and provides a new method for quick, accurate, high-flux and quantitative detection of transgenosis.

Description

technical field [0001] The invention belongs to the technical field of food quality and safety detection, and in particular relates to a combination of primers and probes, a method and a kit for detecting transgenic soybean DAS44406 by double-nested fluorescent PCR. Background technique [0002] Since 1996, when genetically modified crops began to be grown commercially, they have grown at an astonishing rate every year. As of 2014, GM crops have grown to 28 countries in the world, accounting for 60% of the global population, or 4 billion people. In 2015, the planting area of ​​GM crops in the world was 179.7 million hectares, an increase of 100 times compared with 1.7 million hectares in 1996, and GM soybean is the largest GM crop planting area. China is a net exporter of soybeans and soybean products, and rising domestic demand has led to record high imports of genetically modified soybeans. With the large-scale commercialization of genetically modified soybeans, its safe...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6848C12Q2600/16C12Q2531/113C12Q2537/143C12Q2549/113C12Q2549/119C12Q2545/101
Inventor 袁俊杰王莹魏霜马新华龙阳陈文付伟张娜杨卓瑜吴希阳
Owner 中华人民共和国湛江出入境检验检疫局
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com