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

A real-time fluorescence, horse-derived technology, applied in the field of bioengineering, to achieve the effect of high sensitivity, wide application range and simple operation

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

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Experimental program
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Effect test

Embodiment 1

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

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

[0039] The horse chromosome 28 gene (GenBank accession number: NC_009171.2) 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 Software version 3.0 was used to design, and the length of the amplified fragment of the horse-derived component was 125 bp (located in the nucleotide 792-916 interval). The primer and 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 design method of primers and probes, some fragments of the horse chromosome 1 gene sequence (accession number: NC_019458.2) were selected to design primer pairs and probes. The length of the amplified fragment of horse-derived components was 107 bp...

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] Example 3 Sensitivity detection of primer pairs and probes for horse-derived components

[0060] The genomic DNA of the horse 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 the horse sample DNA of 40ng / μl, 20ng / μl, 10ng / μl, 1ng / μl, 0.1ng / μl, 0.01ng / μl, while 0.001ng / μl And 0.0001ng / μl horse sample DNA did not appear the 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 not relat...

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Abstract

The invention discloses a real-time fluorescent PCR detection method for horse-derived components in food and feed, which comprises 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 horse-derived components and quantitatively detect the content of horse-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 horse-derived component and a specific probe for the horse-derived component. The real-time fluorescent PCR detection method of the horse-derived components of the present invention has high specificity, conservatism and high sensitivity, and can avoid the occurrence of false positive results, greatly improving the accuracy of detection, and providing rapid and accurate detection Whether there are horse-derived components in food and feed provides a quantitative detection method, which 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 horse-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 at...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6851C12N15/11
Inventor 王强蔡一村宁雪袁辰刚潘良文
Owner PLANTS & ANIMALS & FOOD TESTING QUARANTINE TECH CENT SHANGHAI ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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