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

A detection kit and technology of source components, applied in the field of detection of quail source components, can solve problems such as research reports on the identification and detection of quail source components that have not yet been seen, and achieve the effect of resisting transmission and spread

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 quail-derived components at home and abroad

Method used

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  • Amplification primer for detecting quail source component by PCR-mtDNA, detecting kit and using method thereof
  • Amplification primer for detecting quail source component by PCR-mtDNA, detecting kit and using method thereof
  • Amplification primer for detecting quail 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 experiment for quail, feed containing quail-derived ingredients, pigeons, chickens, ducks, geese, ostriches, partridges, cattle, sheep, horses, donkeys, and fish animals. Its number is 1, 2kb Marker; DNA;: 2, quail; 3, feed containing quail-derived ingredients; 4, pigeon; 5, chicken; 6, duck; 7, goose; 8, ostrich; 9, partridge; 10. Cattle; 11, sheep; 12, horse; 13, donkey 14, fish; 15. blank control.

[0041] (1) Extraction of total DNA:

[0042] The phenol-chloroform extraction method was used to extract the genomic DNA of quail, feed containing quail-derived ingredients, pigeons, chickens, ducks, geese, ostriches, partridges, cattle, sheep, horses, donkeys, and fish. The results of electrophoresis detection are as follows figure 1 . The purity and concentration of extracted genomic DNA were measured by ultraviolet spectrophotometer. The measured OD260 / OD280 values ​​are both about 1.8, indicating that the DNA purity is high and meets...

Embodiment 2

[0079] Example 2: PCR detection method for feed containing quail-derived ingredients:

[0080] (1) Extract DNA:

[0081] Using phenol-chloroform extraction method: (refer to "Molecular Cloning Test Guide (Second Edition). Beijing: Science Press, 1995: 34-60")

[0082] ①Weigh 0.03g of the ground sample to be tested into a 1.5ml centrifuge tube after autoclaving, and add 500μl of cell lysate for digestion;

[0083] ②Add 10μl of proteinase K (20mg / ml), stir slowly to dissolve and mix, if necessary, add proteinase K again, place it in a water bath at 50℃ for digestion overnight, and mix it several times at the right time;

[0084] ③Cool the cell lysate to room temperature, add an equal volume of Tris saturated phenol, gently rotate the centrifuge tube to mix the two phases, so that the aqueous phase and the phenol form an emulsion;

[0085] ④Centrifuge at 4℃13000rpm / min for 10min, carefully remove the centrifuge tube from the centrifuge, do not shake, it can be seen that there are cle...

Embodiment 3

[0113] Example 3: Preparation of genomic DNA extraction reagent:

[0114] (1) Preparation of cell lysate:

[0115] ①1M Tris-HCl 250mL:

[0116] Weigh 30.285g Tris, add water to make the volume up to 250mL, add HCl to adjust the pH to 8.0, autoclave and store at 4°C.

[0117] ②0.5mol / L EDTA:

[0118] Add 37.22g EDTA-Na in 100mL double distilled water 2 .2H 2 0, Stir vigorously, adjust the pH to 8.0 with NaOH (about 4g NaOH required), dilute to 200mL, autoclave after aliquoting, and store at 4°C

[0119] ③10%(m / v)SDS:

[0120] Dissolve 10g SDS in 90mL double-distilled water, heat to 68°C (to dissolve a few drops of concentrated HCl, pH=7.2), add water to make the volume up to 100mL, separate equipment for use, and store at room temperature without sterilization.

[0121] Take 2 mL of 1 mol / L Tris-HCl (pH 8.0), 40 mL of 0.5 mol / L EDTA (pH 8.0), and 10 mL of 10% (m / v) SDS, and add sterile double-distilled water to make the volume to 200 mL.

[0122] (2) Preparation of 20mg / ml proteina...

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PUM

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Abstract

The invention mainly relates to detection of birds source ingredients, in particular to detection of quail source ingredients. The invention provides a specificity PCR-mtDNA amplification primer used for detecting the quail source ingredients, which is mainly characterized in that: upstream length and downstream length are both 18bp nucleotides; a sequence is as follows: an upstream primer AF (18bp): 5, -TGAGCTCAATAGCCGCCA-3'; a downstream primer AR (18bp): 5'-TGGCTTAATGGTTGGGTG-3'. The invention has the advantages that the invention provides a method and a technique to determine the quail 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 quail 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 present invention mainly relates to the detection of poultry-derived components, in particular to the detection of quail-derived components. Background technique: [0002] The spread of mad cow disease, avian flu, and scrapie disease around the world and cases of infection to humans have aroused the high attention of governments and consumers to the safety of meat and feed. At present, the identification and testing of animal-derived ingredients in livestock and poultry meat products has involved meat industry, import and export trade, markets, and catering industries. At the same time, the inspection and quarantine work in the meat and feed trade markets at home and abroad is increasingly demanding high-tech. Therefore, it is very necessary to develop an accurate, rapid and reliable method to identify animal-derived components of livestock and poultry. [0003] At present, in order to determine the true composition of food and feed, many methods for i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 宗卉张慧霞张利平阮周曦吴建平李丽娟陶虹
Owner SHENZHEN AUDAQUE DATA TECH