Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken

A technology of primers for pigs, cattle and sheep, applied in the field of molecular biology, can solve the problems such as the lack of establishment of a large number of common species with rapid and highly specific primer systems and detection methods, the specificity of PCR reactions cannot be guaranteed, and whether it is a deer product can not be determined. , to achieve the effect of shortening the experimental time, fast, efficient and specific, and convenient to use.

Active Publication Date: 2013-01-16
苏州红冠庄国药股份有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

However, the above scheme does not design primers for deer, so although it can be determined whether the sample contains these four types of substances, it cannot be determined whether it is a deer product
[0005] From the above representative research results, it can be seen that these tests have the following shortcomings: (1) The detection range is narrow, and only a small number of species can be detected at a time, basically no more than four
This is because in the same total DNA template system, the greater the number of primer pairs, the more serious the interference between the primer pairs and the total DNA template, and the less guaranteed the specificity of the PCR reaction; Rapid and highly specific primer systems and detection methods for common species, which can also cause missed detections

Method used

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  • Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken
  • Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken
  • Primer system for PCR (polymerase chain reaction) identification for deer, pig, cow, sheep, horse, donkey, rabbit and chicken

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Experimental program
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Effect test

Embodiment approach 1

[0112] Using the total DNA samples of each species as the total DNA template:

[0113] (1) Treatment of total DNA samples of each species: take 1 μl of total DNA samples from each of the eight animals and dilute them 500 times as total DNA templates, and dilute primers to 1 μmol / L;

[0114] (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.

[0115] Double distilled water 3μl, 10×PCR reaction buffer 1μl, 2.5mM each dNTPs 0.8μl, 2.5U / μl Taq enzyme 0.2μl, (1) the total DNA template 1μl, 1μM forward primer 2μl and 1μM reverse Add 2 μl to the primer; the Taq enzyme is a conventional Taq enzyme (refer to Tiangen’s Taq enzyme manual).

[0116] (3) The formulation of the PCR reaction program: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 45s, annealing at 56°C for 45s, extension at 72°C for 45s, 35 cycles, extension at 72°C for 10 minutes, and finally holding at 4°C A...

Embodiment approach 2

[0129] Using the mixed total DNA sample of the total DNA samples of each species mixed according to a certain ratio as a template:

[0130] (1) Treatment of the mixed total DNA sample: the total DNA samples of the eight animals 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 the mixed total DNA sample and dilute it 500 times as a mixed total DNA template, and dilute the primers to 1 μmol / L;

[0131] (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.

[0132] Double distilled water 3μl, 10×PCR reaction buffer 1μl, 2.5mM each dNTPs 0.8μl, 2.5U / μl Taq enzyme 0.2μl, (1) mixed total DNA template 1μl, 1μM forward primer 2μl and 1μM Reverse primer 2μl; where the Taq enzyme is a conventional Taq enzyme (see Tiangen company's Taq enzyme instructions).

[0133] (3) Formulation of the PCR reaction progr...

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Abstract

The invention discloses a primer system for PCR (polymerase chain reaction) identification for a deer, a pig, a cow, a sheep, a horse, a donkey, a rabbit and a chicken, and can detect eight animal species in one time so as to achieve a purpose that common animal species can be almost covered. The primer of the primer system only carries out specific amplification on a respective species target segment without reacting with other species, and therefore the primer can be suitable for multiple PCR reaction characterized in that at least two primer pairs are induced in one-time PCR reaction so as to effectively shorten experiment time. In addition, when the PCR is carried out, an optimized specific PCR reaction system and reaction program can be used, a detection flow is simplified due to the adoption of a uniform PCR detection method, a quick and efficient identification mode with the high specificity is established so as to effectively identify whether the deer blood product is true and false and identify the adulteration mode, and a modern molecular biology detection means is provided for deer blood quality control. In addition, the primer system can be cooperated with relevant reagent to be prepared into a kit, thereby being convenient to use. Meanwhile, possibility is provided for the industrial production and application, and the primer system has an excellent application prospect.

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, pig, cow, sheep, horse, donkey, rabbit and chicken and a PCR identification method, which is applicable to the rapid detection and identification of the above eight kinds of animal blood products, It is especially suitable for rapid anti-counterfeiting identification of deer blood products. Background technique [0002] Deer blood is the blood of deer family animals. According to the "Compendium of Materia Medica": "Deer blood can nourish deficiency and damage, benefit essence and blood, relieve acne poison and drug poison." As a traditional precious Chinese medicinal material, deer blood is irreplaceable. Its medicinal value has been widely recognized and developed in depth. The product line has been continuously enriched, including deer blood wine, fresh deer blood, deer blood crystal, etc. The market value generated and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 周翠霞杨彦鹏党平
Owner 苏州红冠庄国药股份有限公司
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