Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof

A technology of source components and amplification primers, applied in biochemical equipment and methods, recombinant DNA technology, DNA/RNA fragments, etc. The effect of spreading and spreading

Inactive Publication Date: 2008-09-10
SHENZHEN AUDAQUE DATA TECH
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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 biology methods, there is no research report on the identification and detection of pigeon-derived components at home and abroad.

Method used

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  • Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof
  • Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof
  • Amplification primer for detecting pigeon source component by PCR-mtDNA, detecting kit and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Detection and verification experiments on pigeons; feed containing components derived from pigeons; quail; chickens; ducks; geese; ostriches; partridges; cattle; sheep; horses; donkeys; fish animals.

[0041] (1) Extraction of total DNA:

[0042] Using phenol chloroform extraction method to extract pigeon; feed containing pigeon-derived ingredients; quail; chicken; duck; goose; ostrich; partridge; cattle; sheep; horse; donkey; figure 1 . 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.

[0043] (2) Design of PCR-mtDNA specific primers:

[0044] Compare the mitochondrial gene sequences of various animals published in GenBank, and design PCR-specific primers to identify and detect pigeon-derived components based on their species-conserved sequences (only specific fragments ...

Embodiment 2

[0076] Example 2: PCR detection method for feed containing pigeon-derived components.

[0077] (1) DNA extraction:

[0078] Using phenol-chloroform extraction method: (refer to "Molecular Cloning Test Guide (Second Edition). Beijing: Science Press, 1995: 34-60")

[0079] 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;

[0080] 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;

[0081] 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 the aqueous phase and phenol form an emulsion;

[0082] ④Centrifuge at 13000rpm / min at 4°C for 10min, carefully take out the centrifuge tube from the centrifuge, do not shake, ...

Embodiment 3

[0110] Preparation of Genomic DNA Extraction Reagent:

[0111] (1) Preparation of cell lysate:

[0112] ① 1M Tris-HCl 250mL:

[0113] 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.

[0114] ②0.5mol / L EDTA:

[0115] 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.

[0116] ③10%(m / v)SDS:

[0117] 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.

[0118] Take 1mol / L Tris-HCl (pH8.0) 2mL, 0.5mol / L EDTA (pH8.0) 40mL, 10% (m / v) SDS 10mL, add sterilized double-distilled water to 200mL .

[0119] (2) Preparation of 20 mg / ml proteinase K: 100 mg proteinase K was dissolved in 5 ml sterilized doubl...

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Abstract

The invention mainly relates to detection of bird source ingredients, in particular to detection of pigeon source ingredients. The invention provides a specificity PCR-mtDNA amplification primer used for detecting quail source ingredients, which is mainly characterized in that: upstream length and downstream length are respectively 22bp nucleotides and 20bp nucleotides; a sequence is as follows: an upstream primer GF (22bp): 5'-ACACTGATGCACTTTGTCTTCC-3'; a downstream primer GR (20bp): 5'-TATGTCCTGCGAGCATTCAC-3'. The invention has the advantages that the invention provides a method and a technique to determine the pigeon source ingredients accurately, rapidly and reliably, and has important practical significance to effectively resist spreading bird flu and other epidemic diseases. The invention uses sterilizing ultrapure water to dilute a pigeon DNA template, ensures concentrations to respectively reduce to 12ng/ul,1.2ng/ul,120pg/ul,12pg/ul,1.2pg/ul,120fg/ul,12fg/ul,1.2fg/ul, and then performs PCR amplification. According to an electrophoresis result of the PCR product, the detected minimum concentration is 120pg/ul, namely the sensibility of the primer is 120pg/ul.

Description

Technical field: [0001] The invention mainly relates to the detection of poultry-derived components, in particular to the detection of pigeon-derived components. 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, fast and reliable method for identifying animal-derived ingredients in livestock and poultry. [0003] At present, in order to determine the true ingredients o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 宗卉张慧霞张利平吴建平曹琛福李丽娟
Owner SHENZHEN AUDAQUE DATA TECH
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