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PCR method for tracking source identification of origin components of eight kinds of animals

An animal-derived ingredient, goat technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of no identification method, save manpower and material resources, high specificity and sensitivity, and improve detection efficiency. Effect

Active Publication Date: 2015-09-30
CHINA JILIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the identification of venison and pork can be implemented in national standards and some inventive methods, there is still no efficient identification method for yak meat, venison and camel meat in the current species identification methods

Method used

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  • PCR method for tracking source identification of origin components of eight kinds of animals
  • PCR method for tracking source identification of origin components of eight kinds of animals
  • PCR method for tracking source identification of origin components of eight kinds of animals

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Primer Design

[0032]According to the gene sequence published by GenBank, goat (GU229278), deer (HQ191428), swamp buffalo (AF702618), domestic cattle (EU177861), sheep (KF938337), yak (KM233416), pig (AF486867) and camel (AP003423) The complete mitochondrial DNA (mtDNA) 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, the Primer premier 5.0 software designed universal primers for multiple species. The primers were paired with the mtDNA sequences of the 8 bovine species mentioned above. There were insertion-deletion fragments in the amplified regions under their jurisdiction, so the size of the amplified products of each species was different. In order to improve the detection effect, two downstream primers were designed for optimization at the same time. The primer sequences are a...

Embodiment 2

[0036] Example 2 Optimization of PCR system and reaction conditions

[0037] The PCR reaction system uses a 20 μL basic system, including 2.0 μL of Buffer, 2.0 μL each of primer SEQ ID 1 and optimized primer SEQ ID 2 or SEQ ID 3, 1.6 μL of dNTP mixture, 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.

[0038] According to the above PCR system, a temperature gradient (48.0-65.0° C.) is set on the gradient PCR instrument, and the annealing temperature is optimized first. 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 5 min. After the PCR product was detected by electrophoresis (4S Red agarose nucleic acid dye), the temperature of the electrophoresis band was selected as the temperature of the basic system, at which the size o...

Embodiment 3

[0041] Example 3 PCR product detection and sequence determination

[0042] In the implementation of the present invention, 2.0% agarose gel was used to separate PCR products, and 4S Red nucleic acid dye was added to the melted gel according to the instructions. After 90V constant voltage electrophoresis for 40min, take pictures and observe under the gel imaging system. The results show that the PCR products in the present invention can be cleaned and resolved to determine their size. For specific results, see figure 1 and accompanying drawings.

[0043] The PCR products of each species were recovered according to the operation instructions of the gel recovery kit, and sent to the biological company for sequencing. The sequencing results were compared with the GenBank sequence, and the online software Blast was used for comparative analysis. The similarity and matching degree were both above 98%, indicating that the source of the amplified product was completely consistent wi...

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Abstract

The invention belongs to the technical field of detection of related products of animal origin components including meat and source thereof, and particularly provides a pair of primers capable of detecting the origin components of eight kinds of animals of goat, sheep, buffalo, cattle, pig, deer, camel and yak at the same time in a PCR reaction, the molecular identification technology in the operation process, a kit comprising the primers, and an application method. The nucleotide sequences of the primers are shown in the SEQ ID 1 and the SEQ ID 3. The PCR method is based on the PCR technology, has the advantages of being simple in operation, strong in specificity, low in equipment requirement, and the like, importantly, can detect the origin components of the eight kinds of animals at the same time in once PCR, can be used for adulteration detection of the food meat and the meat products and the tracking source identification of the animal origin components in feed, is simple and quick, and improves the detection efficiency.

Description

technical field [0001] The invention belongs to the technical field of food detection, and in particular relates to a PCR method for identifying 8 kinds of animal-derived components through traceability, that is, using PCR amplification reactions to simultaneously identify the origin of goats, sheep, buffaloes, cattle, pigs, deer, camels and yaks Element. Background technique [0002] Since ancient times, "food is the most important thing for the people", and "food" is also a part of Chinese culture. With the development of social economy and the acceleration of people's life rhythm, the quantity and types of food have undergone great changes. A variety of characteristic meat products in the western region, such as yak meat, desert camel meat and venison, have also been sold to all parts of the country through the Internet. These meats and their various processed products have become an important source and part of people's daily life and leisure food. However, because the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888
Inventor 管峰薛超波李素芳顾佳瑛林昕赵进黄朱梁
Owner CHINA JILIANG UNIV
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