Multiplex PCR (polymerase chain reaction) primer system for synchronously detecting five animal-derived ingredients and detection method

A technology of simultaneous detection of animal-derived components, applied in the field of molecular biology, can solve the problems of long cycle, heavy workload, and incompetent detection technology, and achieve the effect of high specificity and shortened detection time

Pending Publication Date: 2016-11-23
SHANGHAI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the currently promulgated standard methods of conventional PCR-gel electrophoresis and real-time fluorescent PCR can only perform a single qualitative detection of animal-derived meat and meat products, while for processed products with unclear or mixed ingredients, It needs to be determined through multiple detections or multiple detection tests, 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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  • Multiplex PCR (polymerase chain reaction) primer system for synchronously detecting five animal-derived ingredients and detection method
  • Multiplex PCR (polymerase chain reaction) primer system for synchronously detecting five animal-derived ingredients and detection method
  • Multiplex PCR (polymerase chain reaction) primer system for synchronously detecting five animal-derived ingredients and detection method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Under different primer addition amounts of duck species, the total DNA samples of five animals, namely cattle, pigs, sheep, chickens, and ducks, were mixed in equal amounts and diluted to a final DNA concentration of 0.5 ng / μl as sample templates. The multiplex PCR system and reaction conditions of specific primers for pigs, sheep, chickens and ducks were used to amplify the genomes of cattle, pigs, sheep, chickens and ducks respectively, and each reaction was repeated 3 times. Specific steps are as follows:

[0057] 1) Treatment of mixed total DNA samples in equal amounts: After mixing the total DNA samples of 5 animals of cattle, pigs, sheep, chickens, and ducks in equal amounts, dilute to a final DNA concentration of 0.5 ng / μl as sample templates, and Dilute the primers to 10 μmol / L;

[0058] 2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 50 μl / tube. Double distilled water 28-29...

Embodiment 2

[0063] Under different primer addition amounts of chicken species, the total DNA samples of five animals, namely cattle, pigs, sheep, chickens, and ducks, were mixed in equal amounts, diluted to a final DNA concentration of 50 ng / μl, and used as sample templates. The multiplex PCR system and reaction conditions of specific primers for sheep, chicken, and duck were used to amplify the genomes of cattle, pigs, sheep, chickens, and ducks, and each reaction was repeated 3 times. Specific steps are as follows:

[0064] (1) Treatment of equal amounts of mixed total DNA samples: After mixing the total DNA samples of cattle, pigs, sheep, chickens, and ducks in equal amounts, dilute to a final DNA concentration of 50 ng / μl as sample templates, and Dilute the primers to 10 μmol / L;

[0065] (2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 50 μl / tube. Double distilled water 28.8-30.4μl, 10×PCR reactio...

Embodiment 3

[0070] Under different primer addition amounts of pig species, the total DNA samples of five animals, namely cattle, pigs, sheep, chickens, and ducks, were mixed in equal amounts, diluted to a final DNA concentration of 100 ng / μl, and used as sample templates. The multiplex PCR system and reaction conditions of specific primers for sheep, chicken, and duck were used to amplify the genomes of cattle, pigs, sheep, chickens, and ducks, and each reaction was repeated 3 times. Specific steps are as follows:

[0071] (1) Treatment of equal volume mixed total DNA samples: mix the total DNA samples of cattle, pigs, sheep, chickens, and ducks in equal volumes, dilute to a final DNA concentration of 100 ng / μl, and use them as sample templates, and Dilute the primers to 10 μmol / L;

[0072] (2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 50 μl / tube. Double distilled water 29.6-31.2 μl, l0×PCR reactio...

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Abstract

The invention provides a multiplex PCR (polymerase chain reaction) primer system for synchronously detecting five animal-derived ingredients and a detection method. Total DNA (deoxyribonucleic acid) of a sample to be detected is used as a template; a multiplex PCR primer system formed by a primer pair I, a primer pair II, a primer pair III, a primer pair IV and a primer pair V is used for multiplex PCR amplification tests; after the reaction is completed, the results are judged according to agarose gel electrophoresis, wherein the sample total DNA template is one or several of materials in cow, pig, sheep, chicken and duck meat products. When the multiplex PCR primer system provided by the invention is used for detecting the authenticity of the cow, pig, sheep, chicken and duck meat products and the adulteration, the detection speed is high; the specificity is high; the sensitivity is high; the detection efficiency can be greatly improved; the time is saved; in addition, the detection cost is reduced; the multiplex PCR primer system is particularly applicable to the fast anti-counterfeiting identification of cow, pig, sheep, chicken and duck meat products. In addition, the multiplex PCR primer system is matched with relevant reagents and can be made into a kit; the use is convenient; meanwhile, possibility is provided for industrial production and application.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a multiple PCR primer system and a detection method for synchronous detection of five animal-derived components. Background technique [0002] Species identification, species origin and diversity assessment of animals at the nucleic acid molecular level, as well as inspection and research on animal-derived foods, are currently the most effective means for animal-derived components research. Molecular biology methods mainly use DNA analysis technology to determine species according to the DNA sequence in the genome, and can still distinguish animal sources well for thermally processed foods. DNA has more genetic information than protein, and DNA is relatively stable. After autoclaving, DNA fragments of about 300-400bp can still be detected. At the same time, DNA is the basis of molecular biology research, and the separation and purification technology of nucleic acid has also bee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2600/16C12Q2537/143
Inventor 王金斌唐雪明李文王荣谈刘华吴潇蒋玮吕贝贝武国干白蓝
Owner SHANGHAI ACAD OF AGRI SCI
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