Misgurnus anguillicaudatus and paramisgurnus dabryanus specie identification primer and identification method

A technology of paraloach and loach, applied in biochemical equipment and methods, microbe measurement/testing, DNA/RNA fragments, etc., to achieve rapid and efficient germplasm identification and solve species misjudgment effects

Inactive Publication Date: 2015-04-08
中国水产科学研究院北戴河中心实验站
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The present invention aims at the deficiencies of the existing method for discriminating loach and paraloach based on morphology at presen

Method used

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  • Misgurnus anguillicaudatus and paramisgurnus dabryanus specie identification primer and identification method
  • Misgurnus anguillicaudatus and paramisgurnus dabryanus specie identification primer and identification method

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Embodiment 1

[0018] Embodiment 1: The present invention provides a pair of primers based on differences in mitochondrial DNA sequences to identify two species of loach and paraloach. The primer sequences are:

[0019] Upstream primer: 5'-CCGCCCAGGAGTATTTTATG-3' (shown in SEQ ID NO.1);

[0020] Downstream primer: 5'-TGGCATTTCATCAGGGGTG-3' (shown in SEQ ID NO.2).

Embodiment 2

[0022] The present invention provides a method for identifying loach and paraloach based on the primers described in Example 1. In this example, the samples of loach were collected in Zhoushan, Zhejiang, and the samples of paraloach were collected in Qinhuangdao, Hebei. Species identification was carried out according to traditional morphological methods. DNA was extracted according to the conventional phenol / chloroform method, and the collected samples were placed in 400 μL of lysate (NaCl50mmol / L, Tris-Cl (pH=8.0) 30mmol / L, EDTA (pH8.0) 100mmol / L, 1% SDS, Proteinase K 200 μg / mL), incubated at 50°C until clarified, then extracted once with an equal volume of saturated phenol:chloroform:isoamyl alcohol (25:24:1), and then precipitated with an equal volume of isopropanol. Then wash the precipitate with 0.4mL 75% ethanol, dissolve it with TE, and store it at -20°C for future use. The samples were amplified by PCR using the primers described in Example 1. The PCR reaction syste...

Embodiment 3

[0025] The method for identifying loach and paraloach as described in Example 2, the difference is that the samples of loach and paraloach in this embodiment were collected in Panjin, Liaoning, and the PCR amplification product was purified with 8% non-denaturing polymer Acrylamide gel electrophoresis detection, silver staining, scanner scanning record.

[0026] The results showed that a band of 285bp appeared in the 15 samples that were judged as loach by morphology; and a band of 214bp appeared in the 15 samples that were judged by morphology as Paraloach. The identification results of this method are consistent with those based on morphological judgment, which proves the reliability of this method.

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Abstract

The invention provides a molecular biology-based misgurnus anguillicaudatus and paramisgurnus dabryanus specie identification primer and an identification method. The misgurnus anguillicaudatus and paramisgurnus dabryanus specie identification primer is characterized by designing a specific primer sequence: upstream primer sequence being 5'-CCGCCCAGGAGTATTTTATG-3'(shown by SEQ ID NO.1); downstream primer sequence being 5'-TGGCATTTCATCAGGGGTG-3'(shown by SEQ ID NO.2). The identification method comprises the following steps: extracting DNA of misgurnus anguillicaudatus or paramisgurnus dabryanus samples by adopting a conventional method; performing PCR amplification; after the PCR amplification is completed, taking a proper amount of amplified products for performing gel electrophoresis detection to determine the size of amplified fragments, and identifying. The identification method is accurate and high-efficiency. The problems that the traditional morphological distinguishing method is difficult to operate and low in resolution success rate can be solved. Especially, the misgurnus anguillicaudatus and paramisgurnus dabryanu at the young and juvenile stages can be difficulty identified from appearance, and the invention provides a convenient and accurate identification method.

Description

technical field [0001] The invention relates to a molecular biology-based germplasm identification primer and identification method for loach and paraloach, belonging to the technical field of aquatic organisms. Background technique [0002] Paramisgurnus dabryanus belongs to the order Cypriniformes, the family Cobitidae, and the genus Paramisgurnus. Loach (Misgurnus anguillicaudatus) belongs to the order Cypriniformes, the family Cobitidae, and the genus Misgurnus. Paraloach and loach are closely related, similar in shape and often exist in the same domain, and are widely distributed in freshwater waters in my country. In China, both loach and loach are regarded as good products for nourishing and strengthening the body. There is a big gap in market demand, and the market price is rising year by year. In the international market, loach and loach are traditional Chinese foreign trade export commodities, especially popular in Japan, South Korea and China's Hong Kong, Macao ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/166
Inventor 侯吉伦刘永富刘海金王桂兴张晓彦
Owner 中国水产科学研究院北戴河中心实验站
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