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Method, kit and primers for detecting of species of Salmo

A technology for salmon and species, applied in the field of fish detection, can solve the problems of no damage to the fish body, complex operation, strong professionalism, etc., to ensure accuracy and uniqueness, strong repeatability, and high specificity of detection results Effect

Inactive Publication Date: 2014-08-06
SPH NO 1 BIOCHEM & PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the traditional morphological identification method of salmon that requires a complete fish body for comparison of morphological characteristics, and requires a fish body with a complete structure and no damage, which makes the traditional salmon morphological identification method complicated to operate. Defects of strong professionalism

Method used

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  • Method, kit and primers for detecting of species of Salmo
  • Method, kit and primers for detecting of species of Salmo
  • Method, kit and primers for detecting of species of Salmo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 detects the design and synthesis of salmon primers

[0039] 1. According to the official website of NCBI (National Center for Biotechnology Information), search for a total of 445 COI genes of three species of salmon Salmosalar, Oncorhynchus mykiss and Oncorhynchus keta, listed as follows:

[0040] The cytochrome oxidase subunit I (COI) gene of salmon is as follows:

[0041] FJ999166 FJ998966 FJ998729

[0042] FJ999161 FJ998953 FJ998718

[0043] FJ999155 FJ998950 FJ998712

[0044] FJ999148 FJ998803 EU525057

[0045] FJ999131 FJ998799 HQ712702

[0046] FJ999122 FJ998788 HQ611128

[0047] FJ999118 FJ998777 GU440432

[0048] FJ999109 FJ998767 FJ918927

[0049] FJ998992 FJ998756 EF609449

[0050] FJ998986 FJ998745 FJ999539

[0051] FJ998973 FJ998739 FJ999511

[0052] FJ999508 JN007787 EU524353

[0053] FJ999503 JN007784 EU524349

[0054] FJ999485 JN007779 GU324184

[0055] FJ999472 HQ961026 HQ339988

[0056] FJ999469 HQ960962 EU752177

[0057] FJ...

Embodiment 2

[0077] Embodiment 2 detects salmon species

[0078] 1. Extract genomic DNA from the sample

[0079] Take 1g of commercially available salmon meat sample, add liquid nitrogen to grind for 10min, take 0.1g of the crushed sample and add it to a 2ml EP tube, add 1ml of cell lysate, 20μl of proteinase K, and mix well;

[0080] Bath in a constant temperature water bath at 62°C for 20 minutes, centrifuge at 12,000 rpm for 5 minutes in a desktop centrifuge, take the supernatant and carefully transfer it to another centrifuge tube, add 2 times the volume of isopropanol, invert and mix well and you can see filaments, use a 100μl tip pick out, dry

[0081] Add 200 μl TE to rewash and dissolve, add an equal amount of phenol: chloroform: isoamyl alcohol (25:24:1) to shake and mix, and centrifuge at 12,000 rpm for 5 min;

[0082] Take the upper layer solution to another tube, add an equal volume of chloroform:isoamyl alcohol (24:1), shake and mix, and centrifuge at 12000rpm for 5min;

[...

Embodiment 3

[0094] Embodiment 3 detects salmon species

[0095] 1. Extract genomic DNA from the sample

[0096] A salmon tissue sample was taken, and the DNA of the tissue sample was extracted according to the sample gene DNA group extraction method described in Example 2.

[0097] 2. PCR method to amplify the target gene

[0098] (1) PCR reaction

[0099] a. Primers: Take the primer 2 obtained in Example 1, and the forward and reverse primer sequences in the primer 2 are respectively shown in SEQ ID NO:3 and SEQ ID NO:4 in the sequence listing. According to the given concentration of the synthetic primers, a solution with a concentration of 10 μmol / L was prepared for use.

[0100] b. Mg in the PCR reaction system 2+ The concentration is 15mmol / L, the dNTP concentration is 0.3mmol / L, the amplification primer concentration is 0.3μM, the Taq enzyme concentration is 0.1U / μL, and the DNA template concentration is 100ng / μL.

[0101] c. The PCR amplification program is (1) pre-denaturation...

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Abstract

The invention discloses a method, kit and primers for detecting of species of Salmo. The method comprises the following steps: (1) by using genome DNA (deoxyribonucleic acid) of a sample to be detected as a template, amplifying the genome DNA of the sample to be detected by using specific amplification primers, wherein the sequences of the specific amplification primers are respectively disclosed as SEQ ID NO:1-SEQ ID NO:10 in the sequence table; and (2) carrying out gel electrophoresis analysis on the PCR (polymerase chain reaction) amplification product, wherein the sample to be detected with specific strips belongs to Salmo salar or Oncorhynchus keta. Compared with the traditional method, the method disclosed by the invention is accurate and unique. The operating personnel does not need much working experience, and only needs to do the experiment according to the operation steps. The kit has the advantages of short required detection time, low cost, high specificity of detection results and simple detection result judgment method.

Description

technical field [0001] The invention relates to the field of fish detection, in particular to a method for detecting salmon species, a detection kit and primers thereof. Background technique [0002] Salmon are a group of migratory fish that are widely distributed in the Atlantic and Pacific Oceans, and can also be found in continental freshwater lakes. The study found that 90 percent of the salmon that swim into a stream are born in the same stream. Pacific species of salmon typically die within weeks of breeding. Salmon meat, liver, testis and head all have medicinal value. The meat has the effects of tonifying deficiency and fatigue, strengthening the spleen and stomach, warming the stomach, and can treat edema, weight loss, indigestion, fullness, vomiting, acid water, convulsions, and sores. Cod liver oil can be extracted from its liver. Its testis can extract protamine and formulate various protamine preparations, which are suitable for the treatment of reactions ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6869C12Q2565/125C12Q2531/113
Inventor 江文涛黄臻辉
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
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