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Dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously

A real-time fluorescence and rainbow trout technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of salmon aquatic product market supervision, long detection cycle, time-consuming and labor-intensive problems, and achieve enhanced The effect of market supervision, reduced testing cost, and convenient operation

Inactive Publication Date: 2019-03-08
广东省食品检验所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing real-time fluorescent PCR method can only detect one species at a time, which is cumbersome; the ordinary PCR method has a long detection cycle and is time-consuming and laborious
These problems have brought great troubles to the supervision of the salmon aquatic products market

Method used

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  • Dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously
  • Dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously
  • Dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 A dual real-time fluorescent PCR primer set and probe for simultaneous identification of Atlantic salmon and rainbow trout

[0042] The primer set of the present invention can simultaneously detect Atlantic salmon and rainbow trout. The primer set of the present invention includes F1 and R1, F2 and R2, and the base sequences thereof are as follows.

[0043] F1: 5'-agcagaactc agccagcct-3' (SEQ ID NO: 1),

[0044] R1: 5'-aaaggaggga gggagaagtc aa-3' (SEQ ID NO: 2),

[0045] P1: 5'-ccttctggga gatgacc-3' (SEQ ID NO: 3);

[0046] F2: 5'-accatttatta acataaaacc tccag-3' (SEQ ID NO: 4),

[0047]R2: 5'-gtaatgcctgctgccagga-3' (SEQ ID NO: 5),

[0048] P2: 5'-cgtttgagcc gtgcta-3' (SEQ ID NO: 6);

[0049] The above-mentioned F1 and R1 are Atlantic salmon-specific primers, and P1 is an Atlantic salmon-specific probe. The 5' end of P1 is modified by the fluorescent group FAM, and the 3' end of P1 is modified by the non-fluorescent quencher group MGB. F2 and R2 are rainb...

Embodiment 2

[0050] Example 2 A dual real-time fluorescent PCR method for simultaneous identification of Atlantic salmon and rainbow trout

[0051] In this embodiment, four experimental groups are set. Add 50ng of Atlantic salmon DNA to the tube of experimental group 1, add 50ng of rainbow trout DNA to the tube of experimental group 2, add 25ng of Atlantic salmon DNA and 25ng of rainbow trout DNA to the tube of experimental group 3, add Add ddH in 4 2 O served as a negative control.

[0052] 1) Extract sample DNA; weigh the sample with sterile scissors and place it in a 2 mL sterile centrifuge tube, use a commercial animal tissue DNA extraction kit for DNA extraction, and use a nucleic acid protein analyzer to measure the concentration of the extracted DNA.

[0053] 2) Using DNA as a template, using the above-mentioned F1 and R1, F2 and R2 as primers, and P1 and P2 as probes, perform double real-time fluorescent PCR amplification;

[0054] Among them, the dual real-time fluorescent PCR ...

Embodiment 3

[0063] Embodiment 3 specific detection

[0064] In this example, the DNA of king salmon (Oncorhynchus. Tshawytscha) and coho salmon (Oncorhynchus. Kisutch), which are relatively common in the salmon market in China, was used to verify the specificity of the primers. Set up six experimental groups. Add 50ng king salmon DNA to the tube of experimental group 1, add 50ng coho DNA to the tube of experimental group 2, add 25ng Atlantic salmon DNA and 25ng king salmon DNA to the tube of experimental group 3, and add 25ng DNA of king salmon to the tube of experimental group 3. Add 25ng rainbow trout DNA and 25ng king salmon DNA to tube 4, add Atlantic salmon DNA and 25ng coho DNA to tube 5, add rainbow trout DNA and 25ng coho DNA to tube 6 DNA.

[0065] 1) Extract sample DNA: Weigh the sample with sterile scissors and place it in a 2 mL sterile centrifuge tube, use a commercial animal tissue DNA extraction kit for DNA extraction, and use a nucleic acid protein analyzer to measure th...

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Abstract

The invention discloses a dual real-time fluorescence PCR primer group, kit and method for identifying Atlantic salmon and rainbow trout simultaneously. In the invention, two indexes (the Atlantic salmon and the rainbow trout) are detected simultaneously by a pipe-1 PCR, so that the operation is convenient, the detection time is shortened, and the detection cost is lowered. According to the primergroup, the kit and the method, the force of market supervision of salmon aquatic products is intensified, the adulteration behavior in the salmon aquatic products is cracked down, the legitimate interest of consumers is guaranteed, and the powerful technical support is provided.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and specifically relates to a double real-time fluorescent PCR method for simultaneously identifying Atlantic salmon and rainbow trout in salmon aquatic products, which can be used for authenticity identification of salmon aquatic products. Background technique [0002] As one of the world's famous high-value economic fish, salmon meat is fresh and tender, rich in omega-3 unsaturated fatty acids and rich in nutrition. It is a kind of aquatic product loved by Chinese people. The word "salmon" is transliterated from the English "salmon", referring to a commodity name. Worldwide, salmon includes species such as Atlantic salmon, rainbow trout, coho salmon, and king salmon. In the Chinese market, the main species of salmon are Atlantic salmon and rainbow trout. Taxonomically, both Atlantic salmon and rainbow trout belong to the subfamily Salmonidae of the family Salmonidae. At the genus l...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143C12Q2563/107C12Q2561/113
Inventor 周露丁清龙曾晓琮
Owner 广东省食品检验所
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