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Method for quickly identifying collagen and gelatin source in closed tube containing SYBR Green I

A collagen and gelatin technology, applied in biochemical equipment and methods, microbiological measurement/inspection, instruments, etc., can solve the problems of expensive instruments and complicated operation procedures, and achieve simple equipment, simple operation process, and short time Effect

Inactive Publication Date: 2017-10-20
HUAIHAI INST OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the above-mentioned problems of religion and food cost, scholars at home and abroad have successively used chromatography and amino acid composition analysis, polyclonal indirect competitive ELISA, mass spectrometry and species-specific PCR methods to identify the protein source, but the operation process is complicated. , the equipment required for the experiment is more expensive

Method used

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  • Method for quickly identifying collagen and gelatin source in closed tube containing SYBR Green I
  • Method for quickly identifying collagen and gelatin source in closed tube containing SYBR Green I
  • Method for quickly identifying collagen and gelatin source in closed tube containing SYBR Green I

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Cod Collagen Detection:

[0030] (1) Preparation of microcrystalline wax reaction tube: Weigh about 0.05 g of the microcrystalline wax fixed sample and cover it on the liquid surface of the reaction tube containing SYBR Green I dye (0.05-5.00 μl, 1000X), and put the reaction tube into the water bath In the middle, heat up to 60°C, take it out after 10 minutes, and cool and solidify at room temperature.

[0031] (2) DNA extraction: Take 200 mg of cod collagen sample, and use DNeasy mericon Food Kit kit for DNA extraction, and the specific operation steps are carried out according to the kit instructions.

[0032] (3) LAMP reaction: take 5 μl of the extracted DNA, add it to a PCR tube, and add 5.00 μl of 1xThermoPol buffer, MgSO 4 10mM, dNTPs 10mM, Betaine solution 1.0M, 10UBst DNA polymerase, 10μl each of 100umol / L outer primers F3 and B3, 10μl each of 100umol / L inner primers FIP and BIP, 10μl template DNA, 100umol / L loop primer LF and The amount of LB is 10 μl, and st...

Embodiment 2

[0036] Cod gelatin detection:

[0037] (1) Preparation of microcrystalline wax reaction tube: Weigh about 1.00 g of the microcrystalline wax fixed sample and cover it on the liquid surface of the reaction tube containing SYBR Green I dye (0.05-5.00 μl, 1000X), and put the reaction tube into the water bath In the middle, heat up to 100°C, take it out after 10 minutes, and cool and solidify at room temperature.

[0038] (2) DNA extraction: Take 2 g of cod gelatin sample, and use DNeasy mericon Food Kit kit for DNA extraction, and the specific operation steps are carried out according to the kit instructions.

[0039] (3) LAMP reaction: Take 10 μl of the extracted DNA, add it to a PCR tube, and add 1xThermoPol buffer 0.05-μl, MgSO4 5mM, dNTPs 5mM, Betaine solution 0.5M, 5UBst DNA polymerase, 50umol / L outer primer 5 μl each of F3 and B3, 5 μl each of 100 μmol / L internal primers FIP and BIP and 5 μl template DNA, 5 μl of 100 μmol / L loop primers LF and LB, and sterilized water to 1...

Embodiment 3

[0044] Channel catfish collagen detection:

[0045] (1) Preparation of microcrystalline wax reaction tube: Weigh about 1.00 g of the microcrystalline wax fixed sample and cover it on the liquid surface of the reaction tube containing SYBR Green I dye (0.05-5.00 μl, 1000X), and put the reaction tube into the water bath , heated to 100°C, took it out after 8 minutes, and cooled and solidified at room temperature.

[0046] (2) DNA extraction: Take channel catfish collagen sample 100 mg, use DNeasy mericon Food Kit kit for DNA extraction, and the specific operation steps are carried out according to the kit instructions.

[0047] (3) LAMP reaction: take 1 μl of the extracted DNA, add it to a PCR tube, and add 2.5 μl of 1xThermoPol buffer, MgSO4 4mM, dNTPs 2.0mM, Betaine solution 0.2M, 8UBst DNA polymerase, and 10umol / L outer primer 0.625 μl each of F3 and B3, 5 μl each of 10 μmol / L internal primers FIP and BIP, 1 μl template DNA, 0.1-10 μl of 1-100 μmol / L loop primers LF and LB, ...

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Abstract

The invention discloses a method for quickly identifying a collagen and gelatin source in a closed tube containing SYBR Green I. The method comprises the following steps: adding template DNA, a primer, dNTPs, Bst DNA polymerase, MgSO4 and the like into a wax-sealed reaction tube containing SYBR Green I dye on the basis of extracting collagen and gelatin DNA to generate loop-mediated isothermal amplification reaction. According to the method disclosed by the invention, the SYBR Green I dye used in the method is a dye which is combined to all dsDNA double-helix groove areas and has green excitation wavelength; when a DNA sample does not generate special LAMP reaction, the dye is light orange or colorless; when LAMP reaction is generated, the sample shows fluorescent green color; the apparatuses and equipment needed in the whole operation process are simple, influences of sample positive pollution are eliminated, time is short and only lasts for about 2 hours; and the detecting method is economical, is reliable in result, is convenient and quick, and is suitable for base layer quick detection.

Description

technical field [0001] The invention discloses a method for detecting the source of collagen, in particular a method for quickly identifying the source of collagen and gelatin using SYBR Green I closed tube. Background technique [0002] Collagen is a biological polymer substance, and it is the protein with the most abundant content and the largest coverage area in various tissues and organs of mammals. Because of its dual functions of nutrition and physiological regulation, it is widely used in various fields such as medical treatment, food, cosmetics and packaging materials. At present, the commercially available traditional collagen mainly comes from terrestrial animals such as pigs and cattle, but in Islam, non-halal products containing pig gelatin are strictly prohibited, and for bovine collagen, due to the outbreak of foot-and-mouth disease and mad cow disease As a result, its production and sales have also been restricted to a certain extent. In recent years, aquati...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
CPCC12Q1/6844G01N21/6486C12Q2531/119
Inventor 张俊杰王永久段蕊潘霞张博妍
Owner HUAIHAI INST OF TECH