Method for quickly detecting duck-derived components in food

A food and duck source technology, applied in biochemical equipment and methods, material stimulation analysis, microbial measurement/inspection, etc., can solve problems such as inaccurate test results, avoid false negative results, reduce the risk of interference, and operate convenient effect

Active Publication Date: 2013-07-31
南京黄教授食品科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of target gene selection, animal cell nuclear genomic DNA sequences are highly species-specific and can avoid non-specific amplification; in addition, the current research on the detection of animal-derived real-time PCR in food lacks a positive amplification internal standard (IAC) Monitoring the reaction system cannot avoid false negative results, resulting in inaccurate test results
At present, there are no reports on animal-derived fluorescent quantitative PCR detection methods in food that use the DNA sequence of the cell nucleus gene as the target gene and add an internal standard for amplification.

Method used

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  • Method for quickly detecting duck-derived components in food
  • Method for quickly detecting duck-derived components in food
  • Method for quickly detecting duck-derived components in food

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) Sample preparation and DNA extraction

[0024] Operate in accordance with the instructions of the TIANGEN (Tiangen) kit. 50mg of total DNA from samples (duck, chicken, donkey, beef, pork, rabbit, goose, fish, goat, sheep, corn, fennel, soybean, star anise, pepper) were dissolved in 100μL TE buffer , Use a nucleic acid protein analyzer to detect DNA concentration and purity. The results showed that the DNA of these samples met the conditions of fluorescent PCR detection.

[0025] (2) Primer and probe design

[0026] According to the duck (accession number: HQ008784.1) genome sequence (duck IL-2 gene) published in GenBank, the Primer Premier 5.0 software was used to design specific primers and probes, and then the primers and probe sequences were published on the NCBI website Blast analysis comparison and evaluation were carried out to ensure the specificity of primers and probes. The fluorophore FAM is used as the luminescent group of the probe. The primer and p...

Embodiment 2

[0037] 1) The actual detection limit analysis experiment of genomic DNA

[0038] Duck DNA template stock solution (concentration: 250ng / μL) was serially diluted, that is, 250 ng / μL, 25ng / μL, 2.50ng / μL, 0.25ng / μL and 0.025ng / μL 5 concentration gradients, using the optimized reaction system Check its detection limit. The experiment was repeated three times, and three parallels were set up each time, and the Ct value less than 36 was used as the positive criterion.

[0039] The result is as figure 1 As shown, when the template amount in the fluorescent quantitative PCR system is 0.5ng (0.25ng / μL*2μL), there is an obvious amplification curve, and the Ct values ​​of duck DNA are 34.62±0.001. Then the actual detection limit of this fluorescent quantitative PCR detection system is 0.5ng.

[0040] 2) The influence of the amplification internal standard on the actual lower limit of detection

[0041] The minimum detection limit obtained above was used as the amount of target gene t...

Embodiment 3

[0046] Duck DNA from three individual sources with a concentration of 100ng / μL was randomly selected for fluorescent PCR. The experiment was carried out three times, and three parallels were set up each time to investigate the stability of the system. The results are shown in Table 3, and the results show that the standard deviations of the Ct values ​​of the three independent repeated experiments of each sample are all less than 0.3. It shows that the test results have good repeatability and good test stability.

[0047] Table 3 Reproducibility test results of real-time fluorescent quantitative PCR detection method with dual TaqMan probes

[0048]

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Abstract

The invention discloses a method for quickly detecting duck-derived components in food, which is a TaqMan probe fluorescent quantitative PCR (polymerase chain reaction) method. The method comprises the following steps: by using a TaqMan probe containing a DNA (deoxyribonucleic acid) template to be detected, a recombinant plasmid containing positive amplification internal standard DNA and the positive amplification internal standard DNA as well as an IL-2 gene amplification primer and a TaqMan probe for ducks, performing fluorescent quantitative PCR reaction; and performing analysis processing on the results by using ABI7500 Software SDS1.4, wherein the amplification of which the Ct value is less than 36 is taken as a positive detection result. The method firstly proposes that duck-derived components in food can be detected through the fluorescent quantitative PCR method by taking a gene in a karyon as a target gene and adding a competitive amplification internal standard. The method has favorable specificity, can avoid the false negative result by monitoring the PCR reaction in real time, explores a new way to identify animal-derived components in food, and has the advantages of accuracy, stability, convenient operation process and the like.

Description

technical field [0001] The invention belongs to the technical field of food safety detection, and relates to a method for rapidly detecting duck-derived components in food, in particular to a rapid detection method for duck-derived components in food with a TaqMan probe added with an amplified internal standard by real-time fluorescent PCR. Background technique [0002] In recent years, the quality and safety of meat products has become a hot topic of concern to the whole society. Driven by economic interests, some lawbreakers adulterated and sold relatively cheap duck and pork as beef and mutton in order to make huge profits. These behaviors not only damaged the quality of cattle and sheep products, but also caused great harm to Muslims. , it is very likely that the introduction of allergens will endanger the health of consumers, and it will also damage the international reputation of cattle and sheep products. The "horse meat crisis" that occurred in many European countrie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 黄明程欣黄继超周兴虎杨静
Owner 南京黄教授食品科技有限公司
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