A PCR primer and kit for identifying 8 kinds of animal-derived components

A technology of animal-derived components and kits, which is applied in DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of single detection species and poor specificity, and achieve the goal of improving sensitivity, high specificity and sensitivity, and improving detection efficiency. Effect

Active Publication Date: 2018-02-09
ZHOUSHAN INST FOR FOOD & DRUG CONTROL +1
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the deficiency of single detection species and poor specificity in multiple PCR technology in the existing species identification technology, the present invention provides a kind of PCR primer and kit that can identify 8 kinds of animal-derived components in one PCR operation, thereby making up for Insufficiency of existing technology

Method used

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  • A PCR primer and kit for identifying 8 kinds of animal-derived components
  • A PCR primer and kit for identifying 8 kinds of animal-derived components
  • A PCR primer and kit for identifying 8 kinds of animal-derived components

Examples

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Effect test

Embodiment 1

[0024] Example 1 Primer Design

[0025] According to the gene sequence published by GenBank, the mtDNA of goat (GU229278), deer (HQ191428), buffalo (AF702618), cattle (EU177861), sheep (KF938337), yak (KM233416), pig (AF486867) and camel (AP003423) The whole sequence was used as a template, and the mtDNA sequences of 8 species and common species (chicken, duck, rabbit, mouse, horse, dog) were compared and analyzed. According to the specificity and conservation of their gene sequences, Primer premier 5.0 software was used to design A universal primer for multiple species, the primer is paired with the mtDNA sequences of the above-mentioned 8 species of goat, sheep, buffalo, domestic cattle, pig, deer, camel and yak. Moreover, in order to improve the sensitivity of primer detection, the sequence of the upstream primer was modified. The specific primer sequences are shown in Table 1 below.

[0026] Table 1: Primers and sequences in the present invention

[0027]

Embodiment 2

[0028] Example 2 PCR system optimization

[0029] The PCR reaction system uses a 20 μL basic system, including 2.0 μL buffer, 2.0 μL each of primers SEQ ID 1 (or SEQ ID3 or SEQ ID 4) and SEQ ID 2, 1.6 μL of dNTP, 0.2 μL of Taq DNA polymerase, MgCl 2 1.2 μL, DNA template 3.0 μL (about 30ng DNA), ddH 2 O supplemented to 20.0 μL.

[0030] According to the above PCR system, a temperature gradient was set on the gradient PCR instrument to optimize the annealing temperature (temperature range 48.0-63.0° C.). The basic PCR process is as follows: denaturation at 95°C for 5 min; denaturation at 95°C for 40 s, different annealing temperatures for 30 s, and extension at 72°C for 40 s as a cycle, a total of 30 cycles; then extension at 72°C for 10 min.

[0031] The PCR products at different temperatures were detected by electrophoresis (4S Red agarose nucleic acid dye), and the temperature of the electrophoresis band was selected as the temperature of the basic system at which the size ...

Embodiment 3

[0034] Example 3 PCR specificity and sensitivity detection

[0035] In order to verify the specificity and sensitivity of the screening primer combination, amplification reactions were carried out to the genomic DNA comprising the above-mentioned 8 species and chicken, duck, squid, small yellow croaker, horse, donkey, dog, mouse and rabbit, and the upstream primer (SEQ ID NO : 1) and downstream primer (SEQ ID NO: 2) amplification results prove that there is no PCR amplification in other animals except the above 8 species. At the same time, it is also proved that the PCR system has no amplification product for soybean genomic DNA. The primer combinations were then tested for sensitivity. Genomic DNA templates of cattle, sheep, and pigs were diluted to a minimum concentration of 1.0pg / μL according to gradients, and different amounts of DNA templates were added under optimal conditions, and PCR products were detected by electrophoresis after amplification. The results showed th...

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Abstract

The invention provides a PCR primer and kit for identifying eight kinds of animal-derived components. The PCR primer and the PCR kit are capable of simultaneously identifying eight kinds of animal-derived components including goat, sheep, buffalo, domestic cattle, pig, deer, camel and bos grunniens. The nucleotide sequence of the PCR primer provided by the invention is shown in SEQ ID No.1 and SEQ ID No.2. The PCR primer provided by the invention is high in specificity, and low in facility request, can simultaneously detect eight kinds of animal-derived components in the one-time PCR, can be used for adulteration detection of meat and meat products and source tracking identification of animal-derived components, the method is simple, and the detection efficiency is improved.

Description

technical field [0001] The invention relates to a food inspection technology for the identification of animal-derived components in meat and meat products, in particular to a PCR method for synchronous detection of eight animal-derived components in goats, sheep, buffaloes, cattle, pigs, deer, camels and yaks Methods, kits and applications. Background technique [0002] Since ancient times, "food is the most important thing for the people", and "food" is the foundation of survival and an indispensable part of people's daily life. With the development of social economy, meat and its products have become an important source and part of people's daily food. In view of the price and profit of meat and its products, meat adulteration and "baiting" incidents occur from time to time in domestic and foreign markets. Food problems such as "fake beef incident" and "horse meat scandal" not only damage the The interests of consumers have brought many social problems at the same time. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888
Inventor 薛超波管峰顾佳瑛黄朱梁林昕徐爱春
Owner ZHOUSHAN INST FOR FOOD & DRUG CONTROL
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