Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis

A technology of the duck-green channa and channa, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., can solve the problem of ineffective identification of three species of fish, strong subjectivity, etc. problem, to achieve the effect of low cost, obvious difference and strong practicability

Inactive Publication Date: 2014-03-26
NANJING NORMAL UNIVERSITY
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Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of identification primer and method for quick and accurate identification of river snakeheads, duck-green snakeheads and Chinese snakeheads, so as to overcome the existing subjectivity in morphological identification and cannot effectively identify For the defects of the three fish species, the present invention only needs to cut a small amount of caudal fin or other fish body tissue parts for identification without killing the fish species. It has high stability, simple operation, good repeatability, strong practicability and Quick and accurate identification, etc.

Method used

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  • Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis
  • Primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis

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Embodiment

[0018] 1. Extraction and Quantification of Total DNA of River Channa, Yalu Channa and Sino Channa

[0019] 1) Take 0.1-0.3 g of the caudal fin of the experimental fish and add 450 μL of DNA extraction buffer (10 mmol / L Tris-HCl, 5 mmol / L LEDTA, 75 mmol / L NaCl), mix well and then add SDS and proteinase K to the final concentration of 0.5 % and 200 μg / mL, and then digested overnight in a water bath at 55°C.

[0020] 2) Add an equal volume of saturated phenol, phenol / chloroform / isoamyl alcohol (25:24:1), and chloroform / isoamyl alcohol (24:1) to extract once each. Each extraction was mixed gently for 10 minutes, centrifuged at 12000 rpm for 20 minutes at 4°C, and the supernatant was taken.

[0021] 3) Add 2.5mol / L NaCl (final concentration: 0.2mol / L) and pre-cooled 2 times the volume of absolute ethanol to the obtained supernatant for precipitation, centrifuge at 12000rpm for 20 minutes to take the precipitate; rinse with 70% ethanol twice Naturally dry at room temperature, diss...

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Abstract

The invention relates to a primer and method for identifying odontobutis potamophila, o. yaluensis and o. sinensis, particularly relates to identification of odontobutis potamophila, o. yaluensis and o. sinensis by applying microsatellite markers. A pair of primers disclosed by the invention is named as OP34a, and the sequences of the primers are shown in SEQ ID NO. 1 and SEQ ID NO. 2 ( as shown in the Specification). During the identification, the pair of the primers is used to perform PCR amplification on DNA samples of the three types of odontobutis obscura, and then 1.5% sepharose gel (dyed by EB) electrophoresis is used to detect PCR products; the products with 221-251 bp stripes are odontobutis potamophila, the products with 337-351 bp stripes are o. yaluensis, and the products with 465-471 bp stripes are o. sinensis. According to the invention, on the basis of without killing fingerlings, the identification can be performed by shearing a few tail fins or other fish tissue parts only, so that the primer and method have the advantages of high stability, simpleness in operation, excellent repeatability, high practicability, and rapid and accurate identification.

Description

technical field [0001] The invention relates to a method for identifying fish species belonging to the same genus with DNA molecular markers. Specifically, the present invention relates to identifying river snakeheads, duck-green snakeheads and Chinese snakeheads with microsatellite markers. Background technique [0002] Odontobutis potamophila, Odontobutis potamophila, O. yaluensis and O. sinensis belong to Perciformes, Gobies, Odontobutis potamophila, and O. sinensis . The distribution of Channa is quite regular. The local name of Channa in rivers is Channa, Tiger Shark, Nerve, etc. It is only distributed in the middle and lower reaches of the Yangtze River, Qiantang River and Minjiang River systems, and is not found in the Pearl River Basin and the Yangtze River. Upstream, the western limit of the distribution of the Yangtze River system can be traced back to Jingzhou (Shashi), Jiujiang, Huangmei in Hubei, Yingtan, Yichun in Jiangxi and other places. Because of its tende...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 尹绍武张丽娟张国松张亚萍张洪伟胡亚丽
Owner NANJING NORMAL UNIVERSITY
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