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A microdrop PCR primer system and detection method for simultaneous detection of animal-derived components in pigs, sheep and cattle

A detection method and simultaneous detection technology, applied in the field of molecular biology, can solve the problems of single qualitative detection of animal-derived meat and meat products, incompetent detection technology, heavy workload, etc., to shorten and save experimental time , the effect of easy operation

Active Publication Date: 2018-08-31
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These detection standards are currently mainly focused on the use of traditional conventional PCR technology, and can only perform a single qualitative detection of animal-derived meat and meat products
In the actual testing process of meat food samples, for processed products with unclear or mixed ingredients, it needs to go through multiple testing tests to determine, which has a long cycle, heavy workload and high cost.
In the future detection of animal-derived meat and meat products, this type of detection technology has become increasingly incapable of handling the special needs of several or even a dozen animal-derived products at a time, especially when a large number of processed products enter commercial production More efficient and rapid detection methods are needed

Method used

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  • A microdrop PCR primer system and detection method for simultaneous detection of animal-derived components in pigs, sheep and cattle
  • A microdrop PCR primer system and detection method for simultaneous detection of animal-derived components in pigs, sheep and cattle
  • A microdrop PCR primer system and detection method for simultaneous detection of animal-derived components in pigs, sheep and cattle

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Experimental program
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Effect test

Embodiment 1

[0056] The total DNA samples of pigs, sheep, and cattle were mixed according to different combinations, so that the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 500 ng / μl, as a sample template.

[0057] 1) Treatment of mixed total DNA samples: After mixing the total DNA samples of pigs, sheep, and cattle according to different combinations, the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 500 ng / μl, As a sample template, the primers were diluted to 10 μmol / L.

[0058] The different combinations of total DNA samples are: pig, sheep, cow, pig + sheep, pig + cow, sheep + cow, pig + cow + sheep.

[0059] 2) Preparation of the PCR aqueous phase system: Add the following reaction components to a 1.5ml centrifuge tube: 108 μl of double distilled water, 20 μl of 10*PCR reaction buffer, 20 μl of 10mg / mL bovine serum albumin (BSA), and 2.5mMeach of dNTPs 16 μl, 8 μl of 2.5 U / μl Taq enz...

Embodiment 2

[0067] The total DNA samples of pigs, sheep, and cattle were mixed according to different combinations, so that the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 50 ng / μl, and used as sample templates.

[0068] 1) Treatment of mixed total DNA samples: After mixing the total DNA samples of pigs, sheep, and cattle according to different combinations, the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 50 ng / μl, As a sample template, the primers were diluted to 10 μmol / L.

[0069] The different combinations of total DNA samples are: pig, sheep, cow, pig + sheep, pig + cow, sheep + cow, pig + cow + sheep.

[0070] 2) Preparation of the PCR aqueous phase system: Add the following reaction components to a 1.5ml centrifuge tube: 116 μl of double distilled water, 20 μl of 10*PCR reaction buffer, 16 μl of 10 mg / mL bovine serum albumin (BSA), and 2.5 mMeach of dNTPs 12 μl, 9 μl of Taq enzy...

Embodiment 3

[0078] The total DNA samples of pigs, sheep, and cattle were mixed according to different combinations, so that the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 5 ng / μl, and used as sample templates.

[0079] 1) Treatment of mixed total DNA samples: After mixing the total DNA samples of pigs, sheep, and cattle according to different combinations, the final DNA concentration of each animal in different combinations of pigs, sheep, and cattle was 5 ng / μl, As a sample template, the primers were diluted to 10 μmol / L.

[0080] The different combinations of total DNA samples are: pig, sheep, cow, pig + sheep, pig + cow, sheep + cow, pig + cow + sheep.

[0081] 2) Preparation of the PCR aqueous phase system: Add the following reaction components to a 1.5ml centrifuge tube: 90 μl of double distilled water, 20 μl of 10*PCR reaction buffer, 24 μl of 10 mg / mL bovine serum albumin (BSA), and 2.5 mMeach of dNTPs 18 μl, 7 μl of 2.5 U / μl Ta...

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Abstract

The invention discloses a microdrop PCR primer system for synchronous detection of pig, sheet and cattle animal derived components and a detection method. The method includes: taking sample total DNA as the template, using the microdrop PCR primer system composed of a primer pair I, a primer pair II and a primer pair III to conduct microdrop PCR amplification test, and determining the result according to agarose gel electrophoresis at the end of reaction. Specifically, the sample total DNA template is more than one of pig, sheep and cattle species meat products. The invention discloses a rapid, accurate and sensitive method for microdrop PCR synchronous detection of 3 animal derived components, the method can effectively shorten the experimental time, greatly improve efficiency, and save cost, thereby effectively identifying the authenticity and adulteration of pig, sheet and cattle products. In addition, the microdrop PCR primer system designed by the invention cooperates with related reagents to be made into a kit so as to be convenient to use, and at the same time provides possibility for industrial production and application.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a microdroplet PCR primer system and a detection method for synchronously detecting animal-derived components of pigs, sheep and cattle. Background technique [0002] my country is the largest producer and consumer of animal-derived food in the world. The 2013 National Economic and Social Development Statistical Bulletin shows that the annual output of pork, beef, mutton and poultry meat was 83.73 million tons, an increase of 1.8% over the previous year, of which pork output was 54.93 million tons. my country is a big pork production and consumption country, but it is difficult for pork products to enter the international market. In order to ensure the authenticity of food ingredients, the "Twelfth Five-Year Plan" for quality inspection clearly states that it is necessary to focus on strengthening the research on food adulteration identification technology. Among them, the adul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2531/113C12Q2563/159
Inventor 王金斌唐雪明李文潘爱虎李鹏刘华吕贝贝吴潇蒋玮贾军伟白蓝
Owner SHANGHAI ACAD OF AGRI SCI