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Primer system for PCR (polymerase chain reaction) identification of deer/cattle/sheep/horse/donkey/pig animal skin tissue DNA (deoxyribonucleic acid)

An animal skin, cattle and sheep technology, applied in the field of molecular biology, can solve the problems of complex DNA components, interference of PCR results, huge differences, etc., to simplify the detection process, shorten the experimental time, and facilitate use.

Inactive Publication Date: 2014-05-14
苏州红冠庄国药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the huge difference in the DNA composition of skin tissue and blood, the red blood cells that account for the vast majority of blood do not contain genomic DNA, and only white blood cells contain a small amount of genomic DNA; while the genomic DNA in skin tissue accounts for the vast majority, so the DNA composition is more complex. Very easy to interfere with PCR results

Method used

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  • Primer system for PCR (polymerase chain reaction) identification of deer/cattle/sheep/horse/donkey/pig animal skin tissue DNA (deoxyribonucleic acid)
  • Primer system for PCR (polymerase chain reaction) identification of deer/cattle/sheep/horse/donkey/pig animal skin tissue DNA (deoxyribonucleic acid)
  • Primer system for PCR (polymerase chain reaction) identification of deer/cattle/sheep/horse/donkey/pig animal skin tissue DNA (deoxyribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach 1

[0084] Using the total DNA samples of each species as the DNA template:

[0085] (1) Treatment of the total DNA samples of each species: take 1 μL of the total DNA samples of each of the six animal skin tissues and use ddH 2 O was diluted 1000 times as the total DNA template, and the primers were used as ddH 2 O diluted to 2 μM;

[0086](2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 10 μL / tube. Contains 4.9 μL of double distilled water, 1 μL of 10×PCR reaction buffer, 1 μL of 2mM each dNTPs, 0.1 μL of hot-start high-efficiency Taq enzyme at 2.5 U / μL, 1 μL of the total DNA template described in (1), 1 μL of 2 μM forward primer and 1 μL of 2 μM reverse primer; the Taq enzyme is a hot-start high-efficiency Taq enzyme -Blend Taq-Plus- (see TOYOBO company instructions).

[0087] (3) The formulation of the PCR reaction program: pre-denaturation at 94°C for 3min, denaturation at 94°C for 30s, ...

Embodiment approach 2

[0098] Take the mixed total DNA sample after mixing the total DNA samples of each testa tissue according to a certain ratio as a template:

[0099] (1) Treatment of the mixed total DNA sample: the total DNA samples of the six kinds of animal skin tissues were mixed in equal volumes, each of which accounted for 12.5 vol.%, and the resulting mixed total DNA sample was obtained. Take 1 μL of this pooled total DNA sample and wash with ddH 2 O was diluted 1000 times as a mixed total DNA template;

[0100] (2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 10 μL / tube. Contains 4.9 μL of double distilled water, 1 μL of 10×PCR reaction buffer, 1 μL of 2mM each dNTPs, 0.1 μL of 2.5U / μL hot-start high-efficiency Taq enzyme, 1 μL of the mixed total DNA template described in (1), 2 μM forward primer 1 μL and 1 μL of 2 μM reverse primer; the Taq enzyme is a hot-start high-efficiency Taq enzyme-Blend Taq-...

Embodiment approach 3

[0105] Extract the total DNA of deerskin glue with the aforementioned method for extracting total DNA from animal skin tissue, and use it as a DNA template;

[0106] (1) Treatment of DNA samples: The total DNA samples of deerskin glue were treated with ddH 2 O was diluted 1000 times as a mixed total DNA template;

[0107] (2) Preparation of the reaction system: Add the following reaction components into the PCR thin-walled tube, and the reaction system is 10 μL / tube. Contains 4.9 μL of double distilled water, 1 μL of 10×PCR reaction buffer, 1 μL of 2mM each dNTPs, 0.1 μL of hot-start high-efficiency Taq enzyme at 2.5 U / μL, 1 μL of deerskin glue total DNA template described in (1), 2 μM positive 1 μL of the primer and 1 μL of the 2 μM reverse primer; the Taq enzyme is a hot-start high-efficiency Taq enzyme-Blend Taq-Plus- (see the instruction manual of TOYOBO).

[0108] (3) Formulation of the PCR reaction program: the reaction program is the same as that in Example 1.

[010...

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Abstract

The invention discloses a primer system for PCR (polymerase chain reaction) identification of deer / cattle / sheep / horse / donkey / pig animal skin tissue DNA (deoxyribonucleic acid). The primer system can detect six animal species once, and an aim of basically covering common animal species is achieved; primers disclosed by the invention only perform specific amplification on the target fragment of each species without reacting with other species, thus being applicable to the multiplex PCR of introducing two or more primer pairs into the PCR once so as to effectively shorten the experimental time; moreover, when in PCR, by adopting the optimized specific PCR system and reaction process as well as a uniform PCR detection method, the detection flow is simplified, and a fast and efficient identification mode with high specificity is established to effectively identify the authenticity and adulteration conditions of the deer skin / deer skin products, thus a detection means of modern molecular biology is provided for the quality control on the deer skin products; moreover, the primer system also can be made into a kit in combination with related reagent to facilitate the use while making the industrial production and application possible; the application prospects of the primer system are great.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a primer system for PCR identification of deer, cattle, sheep, horse, donkey and pig animal skin tissue DNA and a PCR identification method, as well as a corresponding kit, which is suitable for the above six animal skin tissues The rapid detection and identification of its products are especially suitable for the rapid anti-counterfeiting identification of buckskin and buckskin glue. Background technique [0002] Buckskin is a traditional Chinese medicine in my country with a long history of medicinal use. As early as in "Compendium of Materia Medica", there is a record of deerskin's "invigorating qi, astringent essence, and suppressing sores"; "Sichuan Traditional Chinese Medicine" also records that it "can invigorate qi, astringent and slippery, and treat women's leucorrhea and blood collapse. , Kidney deficiency and Huajing, apply to all sores." Deerskin glue, which...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686
Inventor 党平宋平王瑜杜雨威
Owner 苏州红冠庄国药股份有限公司
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