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Primers for identifying fish-derived components and identification method thereof

A technology based on sourced ingredients and fish, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems that easily cause diarrhea and endanger consumers' health, and reduce the cost of experimental testing , to achieve quantitative detection, good specificity effect

Active Publication Date: 2020-10-09
CHINA MEAT RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Oily fish (Lepidocybium flavobrunneum) looks like cod and is often used as cod, but its muscles and viscera are rich in waxy oil, which can easily cause diarrhea and endanger the health of consumers

Method used

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  • Primers for identifying fish-derived components and identification method thereof
  • Primers for identifying fish-derived components and identification method thereof
  • Primers for identifying fish-derived components and identification method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0055] The preparation method of the template is as follows: mix fish or fish products with sterilized deionized double-distilled water at a weight ratio of 1:3 to 1:5, and use a tissue homogenizer at 10,000 to 14,000 r / min to homogenize for 5 to 10 minutes to prepare Tissue homogenate. Extract total genomic DNA from the sample using a tissue DNA extraction kit.

[0056] Further, in the RT-PCR method, the PCR reaction system uses 25 μL, which contains the following components:

[0057]

[0058] The RT-PCR reaction conditions were: 95°C for 5 minutes; 95°C for 10-15s, 55-60°C for 45-60s, 35 cycles.

[0059] The melting curve preparation conditions are: 95°C for 1 min, 65-70°C for 1 min, and the temperature is raised to 95°C at a rate of 0.02-0.05°C / s. At the same time, the fluorescence intensity was continuously detected; the temperature was lowered to 40°C and kept for 60s, and the first-order negative derivative of the fluorescence signal with respect to the temperature ...

Embodiment 1 3

[0064] The specificity of the primer system of embodiment 1 triple RT-PCR

[0065] 1. Design and synthesis of primers

[0066] Sequencing combined with the NCBI database to obtain 6 species of tuna, 8 species of cod, cod, oil fish (Spiritus mackerel), salmon, yellow croaker, grass carp, silver carp, bighead carp, carp, herring, mackerel, mackerel, sword Based on the mitochondrial DNA sequence information of 260 samples of 26 species of fish such as swordfish, through sequence comparison, 48 primer combinations with good intra-species conservation, good inter-species specificity, and large Tm value differences were found (blue fin 3 pairs of tuna-specific primers, 4 pairs of cod-specific primers, and 4 pairs of oily fish-specific primers), after the evaluation of the Oligo 7.56 primer system, the BLAST on NCBI verified the specificity of the primers, and further screened 8 pairs of candidate combinations (blue 2 pairs of fin tuna primers, 2 pairs of silver cod primers, and 2 p...

Embodiment 2

[0109] Example 2 Sensitivity test for identifying fish species-derived components method

[0110] The muscles of bluefin tuna, cod, and oily fish were taken, and the DNA of the three meats were extracted respectively, and the DNA concentration and purity were calculated. Dilute the three DNA sample concentrations to 5ng / μL, and then serially dilute to 10 4 ,spare. Detection was performed with reference to RT-PCR in Example 1, and a melting curve was prepared. see results Figure 3 ~ Figure 5 .

[0111] Depend on Figure 3 ~ Figure 5 It can be seen that, adopting the multiplex RT-PCR amplification method of the present invention and analyzing the melting peak figure, bluefin tuna DNA dilution 10 4 After doubling, the oily fish DNA was diluted 10 4 After doubling, cod DNA was diluted 10 4 After 10 times, the melting peak can still appear. Therefore, the detection sensitivity of this method for bluefin tuna, cod, and oily fish-derived components can reach picogram level. ...

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Abstract

The invention discloses primers for identifying fish-derived components and an identification method thereof. The invention provides three pairs of specific primers (SEQ ID NO: 1-6), based on multiplereal-time fluorescence PCR melting curves, whether fishes or fish products contain three animal-derived components of blue-fin tuna, silver cod and oil fish or not is identified. The method has the advantages of being accurate in detection, strong in specificity, high in sensitivity, simple, convenient and rapid, can meet the requirement of rapidly identifying whether fishes or fish meat productscontain tuna ventricosa, silver cod and oil fish derived components in large batches, and has huge market requirements and wide application prospects.

Description

technical field [0001] The invention belongs to the technical field of food detection, and in particular relates to a primer and an identification method for identifying fish-derived components. Background technique [0002] With the improvement of people's living standards, aquatic food is more and more popular among consumers, especially the consumption demand of tuna and cod with higher nutritional value is constantly increasing. Tuna is well-known in the international market for its high nutritional value, pure natural, and pollution-free, and enjoys the title of "Ocean Gold". Dicosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), tyrosine, taurine, nucleic acid, iron, calcium, potassium, a variety of essential amino acids and vitamin B12 and other nutrients, eating more can promote Children's brain, bone, and tooth development can prevent and assist in the treatment of iron deficiency anemia; can promote metabolism and delay aging, delay memory decline, and can preve...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2537/143C12Q2527/107C12Q2563/107C12Q2561/113Y02A40/81
Inventor 王守伟许随根李家鹏李金春熊苏玥韦忆萱刘睿茜杨君娜陈曦曲超乔晓玲
Owner CHINA MEAT RES CENT
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