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Rapid detection kit and detection method for carrot components based on loop-mediated isothermal amplification

A loop-mediated isothermal, carrot technology is applied in recombinant DNA technology, microbial assay/inspection, biochemical equipment and methods, etc. specific effect

Inactive Publication Date: 2014-08-20
山东众合天成检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although they are highly sensitive and can detect and amplify nucleic acid samples with less than 10 copies, they also have their own shortcomings that need to be overcome
Technical requirements, material and instrument requirements, and specific defects of the technology itself have seriously restricted the popularization and application of these technologies.

Method used

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  • Rapid detection kit and detection method for carrot components based on loop-mediated isothermal amplification
  • Rapid detection kit and detection method for carrot components based on loop-mediated isothermal amplification
  • Rapid detection kit and detection method for carrot components based on loop-mediated isothermal amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Follow the procedure below for testing:

[0036] (1) Extraction of DNA from non-carrot samples

[0037] A. Weigh 0.1g sample and transfer it to a 1.5mL centrifuge tube. Add 600 μL of preheated lysis buffer, mix gently, and keep warm in a water bath at 65°C for 10 minutes;

[0038] B. Add an equal volume of 600 μL of phenol / chloroform into the tube, mix well by inverting up and down, and extract for 2 minutes;

[0039] C. Centrifuge at 12000g for 5min, draw the supernatant into a new centrifuge tube, add the same volume of sedimentation solution as the supernatant, mix well, let stand at room temperature for 10min, centrifuge at 12000g for 5min, remove the supernatant, and keep the precipitate;

[0040] D. Add 60 μL RNase to the precipitate, place it at 37°C for 2 minutes, mix it well with a pipette tip, dissolve the precipitate at 37°C, add 300 μL buffer solution after 5 minutes, and mix up and down 10 times;

[0041] E. Take out the spin column, place the spin colum...

Embodiment 2

[0054] Follow the procedure below for testing:

[0055] (1) Extraction of DNA from carrot samples with different contents

[0056] A. Add liquid nitrogen to carrots and soybeans, grind them into powder, weigh 0.1 g each, and transfer to a 1.5mL centrifuge tube. Add 600 μL of preheated lysis buffer, mix gently, and keep warm in a water bath at 65°C for 10 minutes;

[0057] B. Add an equal volume of 600 μL of phenol / chloroform into the tube, mix well by inverting up and down, and extract for 2 minutes;

[0058] C. Mix the DNA extraction buffers of carrot and soybean samples in proportion (10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%);

[0059] D. Add the same volume of precipitation solution as the supernatant, mix well, let stand at room temperature for 10 minutes, then centrifuge at 12000g for 5 minutes, remove the supernatant, and keep the precipitate;

[0060] E. Add 60 μL RNase to the precipitate, place it at 37°C for 2 minutes, mix it well with a pipette tip, dissolve the prec...

Embodiment 3

[0074] Follow the procedure below for testing:

[0075] (1) DNA extraction of rice flour samples to be tested

[0076] A. Weigh 0.1g of rice flour and transfer to a 1.5mL centrifuge tube. Add 600 μL of preheated lysis buffer, mix gently, and keep warm in a water bath at 65°C for 10 minutes;

[0077] B. Add an equal volume of 600 μL of phenol / chloroform into the tube, mix well by inverting up and down, and extract for 2 minutes;

[0078] C. Centrifuge at 12000g for 5min, draw the supernatant into a new centrifuge tube, add the same volume of sedimentation solution as the supernatant, mix well, let stand at room temperature for 10min, centrifuge at 12000g for 5min, remove the supernatant, and keep the precipitate;

[0079] D. Add 60 μL RNase to the precipitate, place it at 37°C for 2 minutes, mix it well with a pipette tip, dissolve the precipitate at 37°C, add 300 μL buffer solution after 5 minutes, and mix up and down 10 times;

[0080] E. Take out the spin column, place th...

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Abstract

The invention discloses a rapid detection kit and a detection method for carrot components based on loop-mediated isothermal amplification, belonging to the field of qualitative detection technology for plant-derived components in food and beverages. Specifically speaking, the invention provides the kit and the method for detecting carrot components in food and beverages by using loop-mediated isothermal amplification. The kit comprises 10*Bst buffer, dNTP, magnesium sulfate, betaine, an upstream inner primer, a downstream inner primer, an upstream outer primer, a downstream outer primer, an upstream loop primer, a downstream loop primer, Bst DNA polymerase, a developer and positive control, wherein the 10*Bst buffer contains trihydroxy methyl aminomethane, potassium chloride, ammonium sulfate, magnesium sulfate and Triton X-100. The detection method for carrot components based on loop-mediated isothermal amplification comprises a step of extraction of DNA of food and beverages, a step of loop-mediated isothermal amplification of carrot components and result determination. The kit has the advantages of rapidness, strong specificity, simple operation and good repeatability, and the method has high sensitivity and can detect carrot components with a content of as low as 0.001%.

Description

technical field [0001] The invention relates to a rapid detection kit of carrot components based on loop-mediated isothermal amplification technology and a detection method thereof, and belongs to the technical field of rapid detection of plant-derived components by using nucleic acid amplification technology. Background technique [0002] The continuous expansion of global economic integration and trade internationalization is both an opportunity and a challenge for developing China. At present, my country has become the fifth largest exporter of agricultural products in the world, and the import and export trade of agricultural products continues to grow. In order to protect their own economic interests and compete for the international market, developed countries and regions such as the United States, the European Union, and Japan have tried their best to pass various inspection items and strict or even harsh technical standards, and set up trade barriers for imported agr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2545/101
Inventor 孙敏高宏伟肖西志刘彩霞
Owner 山东众合天成检验有限公司
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