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Primer, kit and method for identifying cobitidae fishes

The technology of a loach fish and a kit, which is applied in the field of molecular biology, can solve the problems of large errors in the morphological identification method, and achieve the effects of shortening the identification time and improving the efficiency.

Pending Publication Date: 2021-11-23
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem of large errors in morphological identification methods, the present invention proposes a primer, a kit and a method for identifying Loachidae fishes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1 Primer Design

[0017] Primers were designed according to the consensus sequence of the mitochondria of Loachidae fishes.

[0018] First primer pair:

[0019] 1F: 5'-ACAGACCTACCGAACTTGGTGATA-3' (SEQ ID NO.1)

[0020] 1R: 5'-ATAGAAACTGACCTGGATTNCTCC-3' (SEQ ID NO. 2).

[0021] Second primer pair:

[0022] 2F: 5'-CTGATCCAACATCGAGGTCGTAAA-3' (SEQ ID NO.3)

[0023] 2R: 5'-AAGCAGCCANCTGAACAGAAAGCG-3' (SEQ ID NO. 4).

Embodiment 2

[0024] Embodiment 2 identification method

[0025] Utilize the primers designed in Example 1 to identify Loachidae.

[0026] S1: DNA extraction of Loachidae fish: 1. Take the fin rays preserved in alcohol, cut about 1g of fin rays into a centrifuge tube, wash them twice with distilled water for 1 hour each time, and put the fin rays at 55°C after cleaning 2. Add 10 μl proteinase K and 500 μl HOM buffer to the centrifuge tube, shake it manually and place the centrifuge tube in a 55°C oven until the tissue is completely lysed and the liquid becomes transparent; 3 .After the tissue is completely lysed, absorb 300μl 4.5mol / L NaCl and 200μl chloroform and add it to the centrifuge tube, vortex gently to mix well, and fully lyse for 15min; 5. Add 490 μl of pure isopropanol solution to the supernatant, vortex gently to mix it, then place it in a refrigerator at 4°C, and let it stand for more than 30 minutes; 6. Add pure isopropanol to the supernatant 490 μl of isopropanol solution, ...

Embodiment 3

[0033] S1: DNA extraction from samples: 1. During the natural reproduction detection period of fishes in the Yangtze River in 2020, three species of fish suspected to be Loachidae were caught; 2. Take the fin rays preserved in alcohol, cut about 1g of fin rays into a centrifuge tube, and wash with distilled water Wash twice, each time for 1 hour. After cleaning, put the fin rays in an oven at 55°C and bake until the alcohol evaporates completely; 3. Add 10 μl proteinase K and 500 μl HOM buffer to the centrifuge tube, shake the centrifuge tube by hand Put it in an oven at 55°C until the tissue is completely lysed and the liquid becomes transparent; 4. After the tissue is completely lysed, add 300 μl 4.5mol / L NaCl and 200 μl chloroform to the centrifuge tube, vortex it gently to mix it evenly Fully lyse for 15 minutes; 5. 13000 rpm, centrifuge for 12 minutes, take 700 μl of the supernatant, and add it to a new 1.5ml centrifuge tube; 6. Add 490 μl of pure isopropanol solution to t...

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PUM

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Abstract

The invention discloses a primer, a kit and a method for identifying cobidae fishes. The identification primer comprises a first primer pair and a second primer pair, the upstream and the downstream of the first primer pair respectively have nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, and the upstream and the downstream of the second primer pair respectively have nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4. The method for identifying the cobidae fishes comprises the following steps of, S1, acquiring DNA of a cobidae fish sample; s2, carrying out PCR amplification on the DNA obtained in the S1 by using the first primer pair to obtain a first PCR amplification product; s3, performing nested PCR amplification by using the second primer pair, and obtaining a second PCR amplification product by using the first PCR amplification product as a template; s4, performing agarose gel electrophoresis and gel cutting purification on the second PCR amplification product obtained in S3 to obtain a purified second PCR amplification product; and S5, sequencing the purified second PCR amplification product, comparing mitochondrial gene sequences of the known species of the cobidae fishes in an NCBI database according to a base sequence of a sequencing result, and determining the species of the cobidae fish sample.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer, a kit and a method for identifying loach family fish. Background technique [0002] Loachidae is one of the family of ray-finned fishes Cyprinidae. Freshwater benthic small fish, omnivorous. Most scholars tend to be divided into three subfamilies, that is, the subfamily of loach, the subfamily of loach, and the subfamily of loach. The distribution of loach family fish is also very extensive in my country, and there are many water systems in southern China, so the loach family is relatively concentrated in the south. There are about 18 genera, 100 species and subspecies of loach family in my country. The appearance of the members of Loachidae is very different from that of Cyprinidae compatriots. Most of them have elongated side-flat or cylindrical bodies and flat heads. Mouth small, inferior, upper jaw formed by premaxilla, with a row of lower pharyngeal teeth, witho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686
Inventor 刘明典宋江腾朱峰跃段辛斌刘绍平陈大庆汪登强
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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