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A lamp rapid detection method for wheat high molecular weight glutenin subunit 1dx5

A glutenin subunit, high molecular weight technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of inability to realize on-site operation, fast, accurate, sensitive, etc., and achieve high specificity and good sensitivity , a huge potential effect

Inactive Publication Date: 2019-07-16
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the defect that the existing technology cannot realize on-site operation, fast, accurate and sensitive wheat high molecular weight glutenin subunit 1Dx5, the present invention provides a rapid detection method of LAMP for wheat high molecular weight glutenin subunit 1Dx5, the purpose of which is to provide A Rapid, Accurate, and Sensitive LAMP Method for the Detection of Wheat High Molecular Weight Glutenin Subunit 1Dx5 with Convenient Field Operation

Method used

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  • A lamp rapid detection method for wheat high molecular weight glutenin subunit 1dx5
  • A lamp rapid detection method for wheat high molecular weight glutenin subunit 1dx5
  • A lamp rapid detection method for wheat high molecular weight glutenin subunit 1dx5

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Embodiment 1

[0032] A rapid detection method for LAMP of wheat high molecular weight glutenin subunit 1Dx5, comprising the steps of:

[0033] a) Design primers, the primer sequence is:

[0034]

[0035]b) Extraction of wheat genome: Take 3-5g of wheat seedlings in the field, wash them, freeze them in liquid nitrogen, and use Cwbiotech Plant Genome DNA Extraction Kit to extract the genome of wheat seedlings after grinding. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value between 1.7 and 1.9 were stored at -20°C for later use.

[0036] c) LAMP amplification: the genome obtained in step b) is subjected to LAMP amplification, the LAMP system is set to a reaction temperature gradient of 60°C, and the total system is 25 μL: among them, primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4) , F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio is 0.8μM:0.8μM:0.1μM:0.1μM, 4mM MgSO4, 1.6mM dNTPs, 0.8M betai...

Embodiment 2

[0041] A rapid detection method for LAMP of wheat high molecular weight glutenin subunit 1Dx5, comprising the steps of:

[0042] a) Design primers, the primer sequence is:

[0043]

[0044] b) Extraction of wheat genome: Take 3-5g of wheat seedlings in the field, wash them, freeze them in liquid nitrogen, and use Cwbiotech Plant Genome DNA Extraction Kit to extract the genome of wheat seedlings after grinding. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value between 1.7 and 1.9 were stored at -20°C for later use.

[0045] c) LAMP amplification: the genome obtained in step b) is subjected to LAMP amplification, the LAMP system is set to a reaction temperature gradient of 61°C, and the total system is 25 μL: among them, primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4) , F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio is 0.8μM:0.8μM:0.1μM:0.1μM, 4mM MgSO4, 1.6mM dNTPs, 0.8M beta...

Embodiment 3

[0049] A rapid detection method for LAMP of wheat high molecular weight glutenin subunit 1Dx5, comprising the steps of:

[0050] a) Design primers, the primer sequence is:

[0051]

[0052] b) Extraction of wheat genome: Take 3-5g of wheat seedlings in the field, wash them, freeze them in liquid nitrogen, and use Cwbiotech Plant Genome DNA Extraction Kit to extract the genome of wheat seedlings. Ultraviolet spectrophotometer Nanodrop 2000 was used to detect the quality and concentration of DNA extraction. DNA samples with OD260 / OD280 detection value between 1.7 and 1.9 were stored at -20°C for later use.

[0053] c) LAMP amplification: the genome obtained in step b) is subjected to LAMP amplification, the LAMP system is set to a reaction temperature gradient of 62°C, and the total system is 25 μL: among them, primers FIP (SEQ ID NO: 3), BIP (SEQ ID NO: 4) , F3 (SEQ ID NO:1), B3 (SEQ ID NO:2) ratio is 0.6μM:0.6μM:0.1μM:0.1μM, 4mM MgSO4, 1.6mM dNTPs, 0.8M betaine (Sigma-Aldr...

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Abstract

The invention provides an LAMP rapid detection method for a high molecular weight glutelin subunit 1Dx5 of triticum aestivum L.. A 1Dx5 gene sequence of the triticum aestivum L. published by GenBank is utilized to design an LAMP primer, an LAMP detection system for the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. is created, and the reaction condition is optimized. Under the condition that the ratios of the LAMP primers FIP, BIP, F3 and B3 are respectively 0.8[mu]m:0.8[mu]m:0.1[mu]m:0.1[mu]m in a 25[mu]L system, the reaction time of 60 minutes and the reaction temperature of 60 DEG C are the optimal reaction conditions, and the LAMP primer can distinguish the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. from the 38 tested triticum aestivum L. materials. The result shows that the created LAMP detection system is simple and rapid, good in sensibility and high in specificity, and the potential on the aspect of detection of the high molecular weight glutelin subunit 1Dx5 of the triticum aestivum L. is huge.

Description

technical field [0001] The invention relates to the technical field of agriculture and plant quarantine, in particular to a LAMP detection method of wheat high molecular weight glutenin subunit 1Dx5 gene. [0002] technical background [0003] Wheat (Triticum aestivum L.) is an important food crop in the world, which can be processed into different foods such as bread, steamed buns, and noodles. The content and quality of protein in wheat is one of the most important indicators affecting the quality of wheat processing, especially glutenin and gliadin, which are the two proteins with the highest content in wheat protein and the greatest impact on wheat processed foods. Glutenin is further divided into high-molecular-weight glutenin subunits and low-molecular-weight glutenin subunits. The gene encoding high-molecular-weight glutenin subunits is located in the homologous group of the first chromosome. In theory, common wheat has six high-molecular-weight glutenin subunits Gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 张小村孔令让杜旭烨
Owner SHANDONG AGRICULTURAL UNIVERSITY