Real-time fluorescent PCR detection method for donkey-derived components in food and feed

A real-time fluorescent, donkey-derived technology, applied in the field of bioengineering, to achieve the effects of good specificity, good intraspecific conservation, and wide application range

Active Publication Date: 2021-09-21
PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The primary purpose of the present invention is to provide a real-time fluorescent PCR detection method for donkey-derived components in food and feed, so as to overcome the shortcomings of the prior art that are prone to false positive results, etc.

Method used

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  • Real-time fluorescent PCR detection method for donkey-derived components in food and feed
  • Real-time fluorescent PCR detection method for donkey-derived components in food and feed
  • Real-time fluorescent PCR detection method for donkey-derived components in food and feed

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Experimental program
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Embodiment 1

[0037] The design of embodiment 1 primer pair and probe

[0038] (1) Design of the first set of primer probes

[0039] The donkey genome scaffold gene (GenBank: NW_014638576.1) was selected as the target gene to design primers and probes. First use NCBI to analyze the sequence and then use Applied Biosystems (ABI; Foster City, CA, USA) company's Primer Designed with Software version 3.0, the length of the amplified fragment for donkey-derived components is 95 bp (located in the nucleotide 581564-581658 interval), and the primer probe sequences are shown in Table 1.

[0040] Table 1 The first set of primer probe sequences

[0041]

[0042] (2) Design of the second set of primer probes

[0043] According to the primer probe design method, some fragments of the donkey genome scaffold gene (GenBank: NW_014638576.1) were selected to design primer pairs and probes. The length of the amplified fragment of donkey-derived components was 108 bp. The primer and probe sequences are...

Embodiment 2

[0052] The preparation of embodiment 2 samples, the extraction of DNA and real-time fluorescent PCR test

[0053] (1) Sample preparation

[0054] Buy fresh lean meat and other commercial meat products from slaughterhouses and supermarkets. They were chopped separately, dried in an oven (UFE500AO; Memeert, Germany) at 80°C for 72 hours, and then pulverized into ultrafine powders using a liquid nitrogen pulverizer (SPEX SamplePrep, USA).

[0055] (2) Extraction of DNA

[0056]Use phenol-chloroform DNA extraction method to extract 100 mg of sample: add 800 μl Tiangen lysate and 10 μl proteinase K, vortex and shake to mix, 65 ° C water bath for 60 minutes, add an equal volume of phenol-chloroform and centrifuge at 12000 rpm for 10 minutes, take The supernatant was purified once more with phenol-chloroform, and then 1 / 10 volume of 3M sodium acetate (pH 5.2) was added. Subsequently, two volumes of ice-bathed absolute ethanol was used for precipitation at -20°C for 30 min, and the...

Embodiment 3

[0059] The primer pair of embodiment 3 donkey-derived components and the sensitivity detection of probe

[0060] The genomic DNA of the donkey was extracted by the DNA extraction method in Example 2, and diluted into 40ng / μl, 20ng / μl, 10ng / μl, 1ng / μl, 0.1ng / μl, 0.01ng / μl, 0.001ng / μl and 0.0001 ng / μl, and then carry out real-time fluorescent PCR detection with the first set of primer probes and the second set of primer probes in Example 1, and the experiment was repeated 6 times. The result is as figure 1 and figure 2 shown.

[0061] figure 1 The results showed that in the 6 tests, amplification curves appeared in donkey sample DNA at 40ng / μl, 20ng / μl, 10ng / μl, 1ng / μl, 0.1ng / μl, and 0.01ng / μl, while 0.001ng / μl And 0.0001ng / μl donkey sample DNA did not appear amplification curve.

[0062] figure 2 The results showed that in the 6 tests, amplification curves appeared in 40ng / μl, 20ng / μl, 10ng / μl, 1ng / μl, 0.1ng / μl and 0.01ng / μl sample DNA, but the amplification curve was n...

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Abstract

The invention discloses a real-time fluorescent PCR detection method for donkey-derived components in food and feed, comprising the following steps: the first step is to use the DNA of the sample to be tested as a template to perform fluorescent quantitative PCR amplification to obtain PCR amplification products ; The second step is to detect the fluorescent signal of the amplified product; the third step is to determine whether the sample contains donkey-derived components and quantitatively detect the content of donkey-derived components in the sample by the Ct value of the detection result; wherein, for PCR The amplified reaction system contains a pair of specific primers for amplifying the donkey-derived component and a specific probe for the donkey-derived component. The specific primer pair and probe for real-time fluorescent PCR amplification of donkey-derived components of the present invention not only have good specificity, but also have high sensitivity, and provide a quantitative method for quickly and accurately detecting whether donkey-derived components are contained in food and feed The detection method has a good application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a real-time fluorescent PCR detection method for donkey-derived components in food and feed. Background technique [0002] The identification and quantification of meat products play an important role in food safety monitoring. In some European countries, all meat products need to clearly label animal-derived ingredients and proportions. Even so, meat adulteration still occurs from time to time. Due to factors such as the similar texture of meat, consumers have no way to accurately identify the type of meat, and illegal traders have an opportunity. Although the adulteration of meat does not necessarily affect health, it greatly increases consumption. the distrust of the recipient. Therefore, it is very necessary and urgent to establish a set of accurate and reliable qualitative detection methods. [0003] At present, primers and probes for detecting animal components a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6888
Inventor 王强许镇坚赵丽娜王赢潘良文
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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