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LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic soybean DP-305423 and derived varieties thereof

A technology of genetically modified soybean and detection kit, applied in the field of molecular biology, to achieve the effect of high sensitivity, low probability and high specificity

Active Publication Date: 2012-08-15
广州迪澳生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Loop-Mediated Isothermal Amplification technology (Loop-Mediated Isothermal Amplification, hereinafter referred to as LAMP method) is a gene amplification technology developed by Eiken Chemical Co., Ltd. in Japan around 2000. It is fast, simple, accurate, easy to popularize, With the advantages of safety and reliability, there is currently no kit that applies the LAMP method to the rapid detection of transgenic soybean DP-305423 and its derivatives

Method used

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  • LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic soybean DP-305423 and derived varieties thereof
  • LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic soybean DP-305423 and derived varieties thereof
  • LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic soybean DP-305423 and derived varieties thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Kit containing chromogen and detection method thereof:

[0056] LAMP detection kit for transgenic soybean DP-305423 and its derivatives, including primer solution, reaction solution, DNA polymerase, control and color reagent:

[0057] (1) Primer solution: Contains 5 μM outer primer 1, 5 μM outer primer 2, 40 μM inner primer 1, 40 μM inner primer 2, the four primers are:

[0058] Outer primer 1: CAAAAAGAGAACACGGGTA (SEQ ID No: 1)

[0059] Outer primer 2: GTATGAGGTGGTGAAGGC (SEQ ID No: 2)

[0060] Internal primer 1: GTGTGAATAAGCAATGTTGGGATCAAGTGGACATACGTGA (SEQ ID No: 3)

[0061] Inner primer 2: AACTAAGAAAGTCTTCCATAGCGAGAAAGAGAAAGAAGATGGT (SEQ ID No: 4)

[0062] (2) Reaction solution: Contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO4 aqueous solution, the volume ratio of the three is 8:5:2;

[0063] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;

[0064] (4) Control: The positive control is the DNA of transgenic soybean DP...

Embodiment 2

[0072] Embodiment 2 The kit and detection method thereof without chromogenic agent:

[0073] The kit is the same as that in Example 1 except that it lacks the chromogen in Example 1.

[0074] Use the above kit to detect the soybean variety to be tested in the following way:

[0075] (1) DNA extraction of the sample to be tested: the DNA of the sample to be tested is extracted and purified by the CTAB method;

[0076] (2) Constant temperature gene amplification reaction: prepare reaction system in 200ul PCR tube: primer solution 1μl, reaction solution 12.5μl, DNA polymerase 1μl, DNA to be tested 2μl, make up to 25μl with sterilized deionized water; set positive control During the reaction, replace the DNA to be tested with the DNA of transgenic soybean DP-305423 at a concentration of 5% or E. coli plasmid DNA containing the target gene. When setting a negative control reaction, replace the DNA to be tested with a reaction mixture that does not contain the target gene; Mix t...

Embodiment 3

[0080] Embodiment 3 PCR reaction and the comparison of detection method sensitivity of the present invention:

[0081] Prepare the LAMP detection kit of transgenic soybean DP-305423 and its derivative varieties according to the following formula:

[0082] (1) Primer solution: Contains 5 μM outer primer 1, 5 μM outer primer 2, 40 μM inner primer 1, 40 μM inner primer 2, the four primers are:

[0083] Outer primer 1: CAAAAAGAGAACACGGGTA (SEQ ID No: 1)

[0084] Outer primer 2: GTATGAGGTGGTGAAGGC (SEQ ID No: 2)

[0085] Internal primer 1: GTGTGAATAAGCAATGTTGGGATCAAGTGGACATACGTGA (SEQ ID No: 3)

[0086] Inner primer 2: AACTAAGAAAGTCTTCCATAGCGAGAAAGAGAAAGAAGATGGT (SEQ ID No: 4)

[0087] (2) Reaction solution: Contains 10mM dNTP, 10×ThermoPol reaction buffer, 150mM MgSO4 aqueous solution, the volume ratio of the three is 8:5:2;

[0088] (3) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;

[0089] (4) Control: The positive control is the DNA of transgenic soybea...

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Abstract

The invention discloses an LAMP (mop-mediated isothermal amplification) detection primer group, kit and method for transgenic soybean DP-305423 and derived varieties thereof. The detection primer group comprises four specific primers. The detection kit comprises a primer solution, a reaction solution, DNA (deoxyribonucleic acid) polymerase, controls and a color developing agent. The detection method is characterized by comprising the following steps: extracting the DNA of the soybean variety to be detected, adopting the four specific primers and the DNA polymerase with strand displacement activity to amplify the sample DNA template at 63-65 DEG C, adding SYBRGreenI to observe color change or observing change of turbidity of the precipitates in a reaction tube with a turbidity meter to judge whether the sample DNA template is amplified and determining whether the soybean variety to be detected contains or is the transgenic soybean DP-305423 and derived varieties thereof, wherein the short-time amplification efficiency can reach 109-1010 copies. The detection primer group, kit and method disclosed by the invention have the advantages of quickness, efficiency, simpleness and convenience in operation and identification, high specificity, high sensitivity, suitability for field detection and the like and are suitable for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method of a transgenic plant variety, in particular to a LAMP detection primer set, a detection kit and a detection method of the transgenic soybean DP-305423 and its derivative varieties. Background technique [0002] soybeans ( Glycine max ) is a leguminous plant whose seeds are rich in protein. Its country of origin is China, and it has been planted for 5,000 years. Since 1996, China has begun to import soybeans in large quantities, and the import volume is equivalent to my country's soybean production. China imports soybeans mainly from the United States, Argentina and Brazil. All three countries are major producers of GM soybeans. Among them, genetically modified soybeans imported from the United States accounted for 89% of the total imports. [0003] DP-305423 soybean was developed by DuPont. The introduced exogenous genes are gm-fad2-1 gene fragment...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 唐大运张隽高苏娟范耀森肖艳文石磊
Owner 广州迪澳生物科技有限公司