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LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method

A technology of genetically modified soybeans and detection methods, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., and can solve problems such as complex reaction systems and operating processes

Inactive Publication Date: 2016-10-26
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction system and operation process of the PCR method are complex and require professionals
In addition, the PCR amplification time is 1.5-2 hours, and the price of the required PCR instrument is around 50,000-100,000 yuan

Method used

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  • LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method
  • LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method
  • LAMP primer group for detecting transgenic soybean GTS 40-3-2 and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The LAMP quick detection method of transgenic soybean GTS40-3-2 comprises the following steps:

[0034] a) Design primers, the primer sequence is:

[0035]

[0036] b) Extraction of soybean genome: 2-3 g of soybean seeds were weighed, ground, and the soybean genome was extracted using a Cwbiotech DNA extraction kit. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value of 1.7-1.9 were selected for LAMP amplification and stored at -20°C for later use.

[0037] c) LAMP amplification: perform LAMP amplification on the genome obtained in step b), and set the reaction temperature gradient of the LAMP system to 61°C; the total system is 25 μL: where primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4 ), F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio of 0.8μM:0.8μM:0.1μM:0.1μM, 4mM MgSO 4 , 1.6mM dNTPs, 0.8M betaine (Sigma-Aldrich, Tokyo, Japan), 1 μL Bst DNA polymerase (New England...

Embodiment 2

[0040] The LAMP quick detection method of transgenic soybean GTS40-3-2 comprises the following steps:

[0041] a) Design primers, the primer sequence is:

[0042]

[0043]

[0044] b) Extraction of soybean genome: 2-3 g of soybean seeds were weighed, ground, and the soybean genome was extracted using a Cwbiotech DNA extraction kit. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value of 1.7-1.9 were selected for LAMP amplification and stored at -20°C for later use.

[0045] c) LAMP amplification: perform LAMP amplification on the genome obtained in step b), and set the reaction temperature gradient of the LAMP system to 63°C; the total system is 25 μL: where primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4 ), F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio of 0.6μM:0.6μM:0.1μM:0.1μM, 4mM MgSO 4 , 1.6mM dNTPs, 0.8M betaine (Sigma-Aldrich, Tokyo, Japan), 1 μL Bst DNA polymerase ...

Embodiment 3

[0048] The LAMP quick detection method of transgenic soybean GTS40-3-2 comprises the following steps:

[0049] a) Design primers, the primer sequence is:

[0050]

[0051] b) Extraction of soybean genome: 2-3 g of soybean seeds were weighed, ground, and the soybean genome was extracted using a Cwbiotech DNA extraction kit. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value of 1.7-1.9 were selected for LAMP amplification and stored at -20°C for later use.

[0052] c) LAMP amplification: perform LAMP amplification on the genome obtained in step b), and set the reaction temperature gradient of the LAMP system to 65°C; the total system is 25 μL: where primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4 ), F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio of 0.8μM:0.8μM:0.1μM:0.1μM, 4mM MgSO 4 , 1.6mM dNTPs, 0.8M betaine (Sigma-Aldrich, Tokyo, Japan), 1 μL Bst DNA polymerase (New England...

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Abstract

The invention relates to an LAMP primer group for detecting transgenic soybean GTS 40-3-2 and a detection method. The method comprises the steps of extracting DNA of a soybean variety to be detected, performing amplification on a template DNA through four designed specific LAMP primers, performing 2 percent agarose gel electrophoresis detection, and determining whether the soybean to be detected is transgenic soybean GTS 40-3-2 by judging whether a strap electrophoresis strip appears or not. According to the method, the DNA amount of each system template is limited to 0.001ng. The primer group has the advantages of high simplicity, high speediness, high specificity and high sensitivity, and a new LAMP detection primer group is provided for the detection of the transgenic soybean GTS 40-3-2.

Description

technical field [0001] The invention relates to the technical fields of agriculture and plant quarantine, in particular to a LAMP detection primer set and a detection method for transgenic soybean Roundup Ready (GTS40-3-2). Background technique [0002] Soybean is one of the most important oil crops in the world. In 2015, soybean oil accounted for about 44% of China's vegetable oil raw materials. my country's annual soybean demand is about 85 million tons. However, my country's soybean production is difficult to meet domestic demand. Since 1996, my country has begun to import a large number of soybeans. In 2014, soybean imports reached 73 million tons, accounting for about 20% of the world's soybean production. At present, the soybeans imported by my country mainly come from the United States, Argentina and Brazil. Among them, the soybeans imported from the United States accounted for about 42% of the total imports in 2014. The U.S. is a big country in the cultivation of ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2531/119
Inventor 张小村许文涛黄昆仑
Owner SHANDONG AGRICULTURAL UNIVERSITY