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A Triple Real-Time Fluorescent PCR Method for Detecting Components of Bovine, Sheep, and Porcine Origin

A porcine-derived, fluorescent technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as difficult to distinguish authenticity, and achieve the effects of saving operating time, multiple freeze-thaw times, and saving reagent costs

Active Publication Date: 2021-04-09
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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  • A Triple Real-Time Fluorescent PCR Method for Detecting Components of Bovine, Sheep, and Porcine Origin
  • A Triple Real-Time Fluorescent PCR Method for Detecting Components of Bovine, Sheep, and Porcine Origin
  • A Triple Real-Time Fluorescent PCR Method for Detecting Components of Bovine, Sheep, and Porcine Origin

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 triple fluorescent PCR method for the detection of components derived from cattle, sheep and pigs has four elements: sample DNA, a pair of universal primers and three probes, fluorescent PCR master mix, and positive control; Aldehyde-3-phosphate dehydrogenase gene design primers and probes to amplify the target sequence and excite and quench the fluorescent signal, but no amplification and fluorescent signal for the non-target sequence; perform fluorescent PCR amplification on the DNA of the sample to be tested The specific methods include: establishing a triple fluorescent PCR primer system for detecting components derived from cattle, sheep and pigs; establishing a triple fluorescent PCR kit for detecting components derived from cattle, sheep and pigs; Determine the triple fluorescent PCR identification method for the detection of bovine, sheep, and porcine-derived components. The invention has the advantages of small standard error, short detection time, simultaneous detection of multiple target genes, and cost saving of reagents; it is suitable for authenticity identification of animal products.

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 Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686
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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