Real-time quantitative PCR method for detecting components of meat product
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A meat product, real-time quantitative technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of inaccurate identification results, and achieve the effect of accurate detection results
Active Publication Date: 2018-10-09
WUHAN POLYTECHNIC UNIVERSITY
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However, there is still the problem of inaccurate identification results in the existing DNA identification methods
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Embodiment 1
[0084] Single component detection of embodiment 1 standard sample
[0085] (1) Preparation of raw materials:
[0086] Take 6 kinds of single-ingredient meat products as standard samples corresponding to the species sources of meat products, numbered 1#~6#, and the source species are respectively pig, cattle, sheep, chicken, duck and rabbit. Take about 1g.
[0087] (2) Genomic DNA extraction:
[0088] Cut pork, beef, mutton, chicken, duck and rabbit meat samples into 1.5mL centrifuge tube and 450μL lysate with sterile scissors, then add 50μL proteinase K, react at 55℃ for 2-4h , repeated blowing several times, centrifuged at 5000rpm for 2min, and took the supernatant; added an appropriate amount of saturated phenol, oscillated and mixed, extracted twice, centrifuged at 5000rpm for 2min, took the supernatant; added 200μL chloroform and extracted again Once, centrifuge at 5000rpm for 2min, take the supernatant; add 0.1 times the volume of 5M NaCl solution, mix well, then add 2...
Embodiment 2
[0108] Example 2 Detection of Meat Products Containing Mixed Components
[0109] (1) Preparation of raw materials:
[0110] Standard mutton, mutton kebab 1, mutton kebab 2 and mutton kebab 3 were used as meat product samples, each taking about 1g, and the preset components to be detected in the meat product samples were mutton, pork and duck.
[0111] (2) By the same method as in step (2) in Example 1, extract a total of 4 groups of genomic DNA from standard mutton, mutton skewers 1, mutton skewers 2 and mutton skewers 3.
[0112] (3) The sequences of 3 pairs of specific primers and 1 pair of 18S rRNA primers required for PCR amplification of the mitochondrial gene NADH oxidoreductase 1 (ND1) are as follows:
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Abstract
The invention discloses a real-time quantitative PCR method for detecting components of meat product. The real-time quantitative PCR method comprises the following steps: extracting genomic DNA of a meat product sample and a standard sample; designing PCR amplification primers required by the genomic DNA of the meat product sample and the standard sample according to the polymorphism difference ofmitochondrial NADH oxidoreductase 1, correspondingly preparing a PCR reaction system for PCR amplification, and reading the Ct value during the amplification process; judging the reliability of PCR amplification according to the contrast between the value Ct of the NADH oxidoreductase 1 in PCR amplification products and the Ct value of 18S rRNA; and judging whether the meat product sample contains any component to be detected according to the difference delta Ct of the Ct value of the NADH oxidoreductase 1 of the genomic DNA amplification products of the meat product sample and the standard sample. The real-time quantitative PCR method can be used for simultaneously detecting one or more of the components with different species sources in the meat product, and has accurate and reliable detection results.
Description
technical field [0001] The invention relates to the technical field of food detection, in particular to a real-time quantitative PCR method for detecting components of meat products. Background technique [0002] For the meat industry, adulteration occurs from time to time, mainly by adding some low-priced meat (such as bamboo rat meat, duck, goose, etc.) to replace some high-priced meat (such as beef, mutton, etc.) Finished product sales. The commonly used meat product identification methods include protein identification (including polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay, liquid chromatography, etc.) and DNA identification (including polymerase chain reaction PCR, RFLP labeling, etc.), among which , DNA identification has the advantage of lower requirements for the type and purity of the material to be identified. However, there is also the problem of inaccurate identification results in the existing DNA identification methods. Contents of...
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