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Triple real-time fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients

A pig-derived, fluorescent technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as difficult to distinguish authenticity, and achieve the effect of saving operation time, short detection time, and small standard error.

Active Publication Date: 2017-08-18
贵州省产品质量检验检测院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] At present, the vast majority of animal products have added flavors, fragrances and flavoring agents, and have been processed. Consumers can only judge the authenticity through observation and sensory evaluation.

Method used

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  • Triple real-time fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients
  • Triple real-time fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients
  • Triple real-time fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 self-made sample

[0037] First, buy beef, mutton, and pork at the local Wal-Mart supermarket, grind them and mix them 1:1:1 for sample preparation, and then extract DNA: use an outsourced DNA extraction kit (name: TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0) to extract Sample DNA; DNA purity is OD 260 / OD 280 =1.81, the DNA concentration was 382.75ng / μL, and stored at -20°C. Afterwards, the DNA was diluted to 100ng / μL for detection. The primer solution, probe solution and positive control were the main components of the kit described in this patent. The negative control and blank control were triple-distilled water. The purchased fluorescent PCR reagent was TaKaRa Premix Ex Taq TM(Probe qPCR), prepared according to Table 3, and set relevant parameters according to Table 4 for on-machine detection (ABI fluorescent PCR instrument, model Step one plus).

[0038] Table 3 reaction system

[0039]

[0040] Table 4 reaction conditions

...

Embodiment 4

[0077] Example 4 Enterprise Entrusted Sample --- Detection of Stir-fried Shredded Pork Pepper

[0078] The list of ingredients includes: rapeseed oil, chili, lean pork, monosodium glutamate, salt, white sugar, pepper. During the process, the purchased DNA extraction kit—Tiangen Deep Processed Food DNA Extraction Kit was used to extract the sample DNA, and the DNA purity was OD 260 / OD 280 =1.80, the DNA concentration was 394.69ng / μL, and stored at -20°C. Afterwards, the DNA was diluted to 100ng / μL for detection. The primer solution, probe solution reagent and positive control were the main components of the kit described in this patent. The negative control was goose-derived DNA, and the blank control was triple-distilled water. The fluorescent PCR reagent was Tiangen Super Real PreMix (Probe), which was prepared according to Table 12, and related parameters were set according to Table 13 for on-machine detection (ABI fluorescent PCR instrument, model 7500Fast).

[0079] Ta...

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Abstract

The invention relates to a triple fluorescence PCR method for detecting bovine-derived, sheep-derived and porcine-derived ingredients. The method comprises four elements, namely a sample DNA, a pair of universal primers and three probes, fluorescence PCR premixed liquid as well as a positive control. The method comprises the following steps: designing primers and probes according to glyceraldehyde-3-phosphate dehydrogenase genes of cows, sheep and goats as well as pigs, amplifying a target sequence, and exciting and quenching a fluorescence signal, wherein a non-target sequence is not amplified and has no fluorescence signal; and preparing a kit according to a formula of a reagent used in fluorescence PCR amplification carried out on a to-be-detected sample DNA and amplification conditions. Concrete actions are as follows: a triple fluorescence PCR primer system used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is established; a triple fluorescence PCR kit used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is established; and a triple fluorescence PCR identification method used for detecting the bovine-derived, sheep-derived and porcine-derived ingredients is determined. The method provided by the invention has the advantages that the standard error is small, the detection time is short, multiple target genes can be detected at the same time, and the reagent cost is saved; and the method is applicable to true and false identification of an animal product.

Description

technical field [0001] The invention relates to an identification method including genes, in particular to a method for detection of origin of animal products and identification of authenticity and falsehood. Background technique [0002] At present, the vast majority of animal products have added flavors, fragrances and flavoring agents, and have been processed. It is difficult for consumers to distinguish the authenticity only through observation and sensory evaluation. [0003] In the research on methods of authenticity and adulteration detection of animal products using molecular biology techniques, amplification and detection of target animal-derived genes by PCR technology is currently the mainstream scientific basis and technical means, and has been widely used in recent years. Conventional PCR technology has the following characteristics in this field: ① DNA can be extracted from most animal products; ② Reasonably designed primer pairs can specifically identify targe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2537/143C12Q2563/107
Inventor 孙端方李春宇田志强董睿寻思颖罗绍楠刘廷菊
Owner 贵州省产品质量检验检测院
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