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Amplification primer for PCR-mtDNA test of total chicken origin

An amplification primer and kit technology, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc., can solve problems such as research reports on the identification and detection of total poultry-derived components, etc. The effect of spreading and spreading

Inactive Publication Date: 2008-09-24
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research on the identification and detection of animal-derived components by molecular biological methods, there is no research report on the identification and detection of total poultry-derived components at home and abroad

Method used

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  • Amplification primer for PCR-mtDNA test of total chicken origin
  • Amplification primer for PCR-mtDNA test of total chicken origin
  • Amplification primer for PCR-mtDNA test of total chicken origin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: For quail, pigeon, chicken, duck, goose, ostrich, partridge, cattle, sheep, horse, donkey, fish animal and containing quail, pigeon, chicken, duck, goose, ostrich, partridge, cattle, sheep , horse, donkey, fish-derived ingredient feed detection verification experiment.

[0044] (1) Extraction of total DNA:

[0045]Use phenol chloroform extraction method to extract the genomic DNA and DNA containing quail, pigeon, chicken, duck, goose, ostrich, Partridge, cattle, sheep, horses, donkeys, and fish-derived ingredients feed DNA, electrophoresis test results (such as figure 1 , 2 ). The purity and concentration of extracted genomic DNA were determined by UV spectrophotometer. The measured OD260 / OD280 values ​​were both about 1.8, indicating that the DNA purity was high and met the requirements of PCR amplification.

[0046] (2) Design of PCR-mtDNA specific primers:

[0047] Compare the mitochondrial gene sequences of various animals published in GenBank, ...

Embodiment 2

[0114] Example 2: PCR detection method for feed containing components of poultry origin.

[0115] The feeds containing quail, pigeon, chicken, duck, goose, ostrich, partridge, cattle, sheep, horse, donkey and fish-derived ingredients were tested respectively.

[0116] (1) DNA extraction:

[0117] Using phenol-chloroform extraction method: (refer to "Molecular Cloning Test Guide (Second Edition). Beijing: Science Press, 1995: 34-60")

[0118] ①Weigh 0.03g of the crushed sample of the substance to be detected into a 1.5ml centrifuge tube after autoclaving, and add 500μl of cell lysate to digest;

[0119] ② Add proteinase K (20mg / ml) 10μl, stir slowly to dissolve and mix well, if necessary, add proteinase K again, put it in a water bath at 50°C for digestion overnight, and mix well several times in due course;

[0120] ③ Cool the cell lysate to room temperature, add an equal volume of Tris-saturated phenol, and gently rotate the centrifuge tube to mix the two phases, so that th...

Embodiment 3

[0149] Preparation of Genomic DNA Extraction Reagent:

[0150] (1) Preparation of cell lysate:

[0151] ① 1M Tris-HCl 250mL:

[0152] Weigh 30.285g Tris, add water to make up to 250mL, add HCl to adjust the pH to 8.0, autoclave, and store at 4°C.

[0153] ②0.5mol / L EDTA:

[0154] Add 37.22g EDTA-Na2.2H2O to 100mL double-distilled water, stir vigorously, adjust the pH to 8.0 with NaOH (about 4gNaOH is needed), set the volume to 200mL, autoclave after aliquoting, and store at 4°C.

[0155] ③10%(m / v)SDS:

[0156] Dissolve 10g of SDS in 90mL of double-distilled water, heat to 68°C (to help dissolve a few drops of concentrated HCl, pH=7.2), add water to make up to 100mL, aliquot and store at room temperature without sterilization.

[0157] Take 2 mL of 1 mol / L Tris-HCl (pH 8.0), 40 mL of 0.5 mol / L EDTA (pH 8.0), 10 mL of 10% (m / v) SDS, add sterilized double distilled water to make up to 200 mL.

[0158](2) Preparation of 20 mg / ml proteinase K: 100 mg proteinase K was dissolved...

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Abstract

The invention mainly relates to detection of poultry-derived component, which mainly relates to detection of poultry-derived component by using PCR-mtDNA. A specific PCR-mtDNA amplification primers for detecting poultry-derived component is characterized by compring a upstream ribonucleotide with length of 18bp, a downstream ribonucleotide with length of 19bp, and the sequence of the upstream primer PF(18bp) is 5'-AGAACTACGAGCACAAAC-3', and the sequence of the downstream primer PR(19bp) is 5'-GCTATACCTTGACCTGTCT-3'. The detection of poultry-derived component by using PCR-mtDNA has the advantages that: the method is an accurate, rapid and reliable identification method and technology of poultry-derived component, and has the important practical significance of effective resistance on propagation and spread of bird flu and other poultry diseases. The research utilizes aseptic ultrapure water to dilute DNA template of poultry to cause the concentration to decrease to 12ng per Mul, 1.2 ng per Mul, 120 pg per Mul, 12 pg per Mul, 1.2 pg per Mul, 120 fg per Mul, 12 fg per Mul and 1.2 fg per Mul, and carries out PCR amplification. Known from the electrophoresis result of PCR product, the minimum concentration which can be detected is 120 pg per Mul, so the sensitivity of the primer is 120 pg per Mul.

Description

Technical field: [0001] The invention mainly relates to the detection of poultry-derived components, and mainly relates to the detection of total poultry-derived components by using PCR-mtDNA. Background technique: [0002] The spread of mad cow disease, avian influenza and scrapie in various parts of the world and cases of infecting humans have caused governments and consumers to pay close attention to the safety of meat and feed. At present, the identification and detection of animal-derived components in livestock and poultry meat products has involved the meat industry, import and export trade, markets, and catering industries. At the same time, the inspection and quarantine work in the domestic and foreign meat food and feed trade markets has an increasingly urgent demand for high-tech. Therefore, it is very necessary to develop an accurate, rapid and reliable method for identifying total poultry-derived components. [0003] At present, in order to determine the authe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 张利平张慧霞宗卉吴建平李丽娟阮周曦
Owner GANSU AGRI UNIV
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