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Method for rapidly detecting LOX (lipoxygenase) transgenic wheat and kit using method

A fast technology for transgenic wheat, which is applied in the field of molecular genetic breeding, can solve the problems of defective technology and technical inspiration, and has not been applied, and achieve the effects of improved recognition, short detection cycle and low detection cost

Active Publication Date: 2017-03-22
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Loop-mediated isothermal amplification technology relies on primers that can recognize 6 specific regions on the target sequence and a DNA polymerase with strand displacement properties, which can efficiently, rapidly and highly specifically amplify the target sequence under isothermal conditions. However, this method has not been applied in the detection of exogenous genes in transgenic wheat. Therefore, establishing a method for detecting LOX transgenic wheat based on LAMP and developing a corresponding detection kit has important popularization and application value, but the defects in the prior art are corresponding technology and technical implications, it cannot be promoted and applied

Method used

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  • Method for rapidly detecting LOX (lipoxygenase) transgenic wheat and kit using method
  • Method for rapidly detecting LOX (lipoxygenase) transgenic wheat and kit using method
  • Method for rapidly detecting LOX (lipoxygenase) transgenic wheat and kit using method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 LAMP detection kit for LOX transgenic wheat lines

[0049] A test kit using the method for detection, which contains:

[0050] 10×LAMP reaction buffer 2.5 μL, concentration 8 U / μL Bst DNA polymerase 1.0 μL, primers F3 and B3 each at 10 μmol / L concentration 0.5 μL, primers BIP and FIP each at 20 μmol / L concentration 1.6 μL, 1.0 μL of DNA template, 4.0 μL of betaine at a concentration of 5 mol / L, 3.5 μL of dNTPs at a concentration of 10 mmol / L, and MgSO at a concentration of 100 mmol / L 4 Solution 1.0 μL, chromogenic reagent hydroxynaphthol blue 1.0 μL, with ddH 2 O was added to 25 μL; the positive control was 100 μL of transgenic wheat DNA solution with a concentration of 1 μg / μL, and the amount of each positive control was 1 μL; the primers included:

[0051] Outer primer F3: 5'-GTTCAGCCAGTGCTGACAA-3' (SEQ ID No.1);

[0052] Outer primer B3: 5'-AGCCTAGCCAATCTTCACAATC-3' (SEQ ID No.2);

[0053] Internal primer FIP: 5'-CGCCTGCAGGTCGACCATATGGCTGATGCCATCTAG ACT...

Embodiment 2

[0055] Example 2 Simple Detection of LOX Transgenic Wheat and Its Derivatives by LAMP

[0056] The kit described in Example 1 was used for operation.

[0057] Take 1 μL of transgenic wheat DNA to be tested as a template, and prepare a 25 μL reaction system according to the kit requirements:

[0058] Including 2.5 μL of LAMP reaction buffer, 1.6 μL of internal primers FIP and BIP, 0.5 μL of external primers F3 and B3, 4.0 μL of betaine, 3.5 μL of dNTPs, 1.0 μL of hydroxynaphthol blue, MgSO 4 solution 1.0 μL and Bst DNA polymerase 1.0 μL with ddH 2 O make up to 25 μL;

[0059] The mixed reaction was incubated at 63°C for 40-60min, and finally heated at 80°C for 5min to terminate the reaction;

[0060] The reaction solution changes from purple blue to sky blue, indicating that the detected sample is LOX transgenic wheat; otherwise, the sample to be tested is non-LOX transgenic wheat (results such as figure 1 shown).

Embodiment 3

[0061] Example 3 LAMP detection of LOX transgenic wheat and its derivatives

[0062] This embodiment is to verify the detection result of embodiment 2 and carry out repeated tests under the same conditions, but according to the kit described in embodiment 1, except for the chromogen;

[0063] Using the DNA of the sample to be tested as a template, prepare a 25 μL reaction system according to the requirements of the kit. The mixed reaction was incubated at 63°C for 40-60min, and finally heated at 80°C for 5min to terminate the reaction. The reactants show characteristic bands through electrophoresis detection, which proves that the detected sample is transgenic wheat (results such as figure 1 shown), by figure 1 with figure 2 It can be seen that the detection result of the present invention completely corresponds to the gel electrophoresis result, and the accuracy is high.

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Abstract

The invention relates to the field of molecular genetic breeding and particularly provides a method for rapidly detecting LOX (lipoxygenase) transgenic wheat and a kit using the method. Specific and sensitive outer primers including F3 and B3 and inner primers including FIP and BIP are taken as amplification target sequences. The principle of the detection method is as follows: if the color of a reaction liquid turns from purplish blue to sky blue through DNA (deoxyribonucleic acid) extraction and LAMP (loop-mediated isothermal amplification) detection, detected wheat belongs to a transgenic wheat strain. The method has low requirement for the quality of a DNA template, the LAMP detection cycle is short, the detection cost is low, the operation process is simple and convenient, and a result is easy to analyze.

Description

technical field [0001] The invention relates to the field of molecular genetic breeding, and specifically provides a method for rapidly detecting LOX transgenic wheat. Background technique [0002] Lipoxygenase (LOX) is one of the main factors affecting the color of wheat flour and flour products and storage properties. Lipoxygenase in wheat grains can oxidize carotenoids and increase the whiteness of flour. At the same time, low lipoxygenase activity can effectively reduce the oxidation reaction of lipids and weaken the phenomenon of oxidative deterioration of grains, thereby improving the storage properties of wheat. Using transgenic technology to carry out the study of wheat translipoxygenase gene, in order to improve the color and storage characteristics of wheat flour, improve the quality of wheat. In order to promote the application of transgenic technology in wheat genetic improvement, the simple and rapid identification of exogenous genes has become the primary pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6895C12Q2600/13C12Q2531/119C12Q2527/125
Inventor 孔令让王彪杜旭烨
Owner SHANDONG AGRICULTURAL UNIVERSITY
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