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One-time rapid identification method of pig, chicken and rabbit meat

A chicken and pork technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of unstable test results, cumbersome operation, and difficulty in distinguishing mixed meat samples, and the number of primers can be reached. Reduced, reduced false positives, significant specificity

Active Publication Date: 2019-08-13
QINGDAO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methods that have been proposed or used, such as infrared spectroscopic detection, protein fingerprinting methods, immunological methods, random amplified polymorphic DNA analysis (RAPD), restriction fragment length polymorphism analysis (RFLP) and amplified fragment length multiple Physiological analysis (AFLP) has some disadvantages, such as unstable test results, difficult to distinguish mixed meat samples, cumbersome operation, etc.

Method used

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  • One-time rapid identification method of pig, chicken and rabbit meat
  • One-time rapid identification method of pig, chicken and rabbit meat
  • One-time rapid identification method of pig, chicken and rabbit meat

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: Screening of amplification primers

[0029] Firstly, the most important primers in the PCR reaction were designed, and the detection sensitivity of the designed primers was verified and screened.

[0030] 1) Primer design

[0031] The mitochondrial cytochrome b gene with obvious differences in specificity between species was selected as the target gene. At the same time, in order to realize the purpose of identifying the three species of chicken, rabbit meat and pork at one time, and reduce the number of primers added in the experiment, first Determine the highly conserved region of the cytochrome b gene of each group of species, then find out the difference region in the highly conserved region of each species, and design a specific primer set in the difference region. Use Oligo 6.0, Primer5.0, DNAMAN and other software to design multiple sets of primers, and the annealing temperature is required to be roughly the same, so that a PCR reaction can amplify 3 ...

Embodiment 2

[0043] 1. Sample preparation and DNA extraction

[0044] Take an appropriate amount of commercially available chicken and rabbit meat, mix them until homogeneous, and randomly cut out 6 portions with clean tweezers or scissors, about 20 mg each, grind them with liquid nitrogen, and place them in six 1.5mL centrifuge tubes. DNA was extracted with an animal tissue whole genome extraction kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). Tissue digestion time is 2-3 hours. The OD of purified DNA was measured by UV spectrophotometer 260 / OD 280 All between 1.8-2.0, the concentration is 20-50ng / μL, 6 tubes of DNA are thoroughly mixed with each other to make a sample.

[0045] 2. PCR amplification

[0046] In a 25 μL PCR reaction system, 2.5 μL of 10-fold PCR buffer, 1 μmol / L of each of the four dNTPs, 2 μL of primer sets for identifying pork, chicken or rabbit meat, 1.25 U of Taq DNA polymerase, 100 ng of genomic DNA, and the rest Sterile distilled water.

[00...

Embodiment 3

[0052] 1. Sample preparation and DNA extraction

[0053] Take an appropriate amount of commercially available pork and add a small amount of chicken and rabbit meat to make dumpling fillings. Use clean tweezers or scissors to randomly cut out 6 portions, about 20 mg each, grind them with liquid nitrogen and place them in 6 1.5mL centrifuge tubes. DNA was extracted with an animal tissue whole genome extraction kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). Tissue digestion time is 2-3 hours. The OD of purified DNA was measured by UV spectrophotometer 260 / OD 280 All between 1.8-2.0, the concentration is 20-50ng / μL, 6 tubes of DNA are thoroughly mixed with each other to make a sample.

[0054] 2. PCR amplification

[0055] In a 25 μL PCR reaction system, 2.5 μL of 10-fold PCR buffer, 1 μmol / L of each of the four dNTPs, 2 μL of primer sets for identifying pork, chicken or rabbit meat, 1.25 U of Taq DNA polymerase, 100 ng of genomic DNA, and the rest Sterile...

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Abstract

The invention provides a method for rapidly identifying pork, chicken and rabbit meat at a time. A unified detection method is needed to identify the fresh pork, chicken and rabbit meat and ingredients of the pork, chicken and rabbit meat in mixed or processed meat products for the phenomenon that chicken and rabbit meat leftovers are processed to counterfeit pork. A traditional detection method is long in detection time, and time and labor are consumed. Mitochondria cytochrome b genes of the pork, chicken and rabbit meat serve as feature target genes to design three pairs of specific primers, namely, F / R pork, F / R chicken and F / R rabbit meat, specific amplification fragments are obtained through PCR, whether target stripes exist in an electrophoretogram or not is analyzed, and then whether meat samples are adulterated or not is detected. The method can accurately, stably, easily and rapidly identify duck meat, the chicken and the rabbit meat and the ingredients of the duck meat, the chicken and the rabbit meat in the mixed or processed meat products.

Description

technical field [0001] The invention belongs to the technical field of food quality and safety detection, and relates to a PCR technology detection method for one-time rapid identification of pig, chicken and rabbit meat. Background technique [0002] my country is a big meat consumption country, and meat is an extremely important category in the food source of Chinese residents. In recent years, with the improvement of people's living standards, the meat market has also expanded rapidly. Driven by illegal traders, adulteration incidents have occurred frequently. At present, the means of adulteration are mainly fake ones. For example, some merchants mix different kinds of meat in their products, such as chicken, rabbit meat and their leftovers to make dumpling fillings, etc. Meat adulteration has become an issue affecting our country. Another big problem of food quality and safety. Therefore, it is very important to quickly and accurately detect meat for adulteration. How...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12Q1/6888C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143
Inventor 孙海新于金鑫孙丕春范忠刚
Owner QINGDAO UNIV
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