Multiple PCR method for rapidly identifying four varieties of sea cucumbers

A multiplex, sea cucumber technology, applied in the field of multiplex PCR for rapid identification of four sea cucumber varieties, can solve the problems of heavy workload and long time, and achieve the effects of improving efficiency, reducing cost and high specificity

Active Publication Date: 2012-10-24
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Advantages of the present invention: 1. Multiplex PCR identification of sea cucumber species overcomes the shortcomings of traditional morphological methods such as large workload and long time consumption when identifying sea cucumbers

Method used

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  • Multiple PCR method for rapidly identifying four varieties of sea cucumbers
  • Multiple PCR method for rapidly identifying four varieties of sea cucumbers
  • Multiple PCR method for rapidly identifying four varieties of sea cucumbers

Examples

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Embodiment

[0014] Identification of Dried Sea Cucumber Species

[0015] (1) Pretreatment of sea cucumber samples: rinse dried sea cucumbers with tap water, soak in distilled water at 55°C overnight;

[0016] (2) Extraction of DNA template: ① Weigh 100 mg of sea cucumber muscle column, wash it, dry it with sterile paper, chop it or place it in liquid nitrogen to grind it. Then add 400 μL of lysis solution (50 mM Tris-HCl pH 7.5; 1.5% CTAB; 1M NaCl; 15 mM EDTA) and 12 μL of 20 mg / mL proteinase K, and place at 55° C. for 5-8 hours until the sample is dissolved. ② Add 600 μL chloroform to the overnight muscle column, mix for 3 minutes, let stand for 7 minutes, and centrifuge at 12000 g for 2 minutes. ③Take the supernatant, add 500μL chloroform, mix by inversion for 2min, and centrifuge at 12000g for 2min. ④ Take the supernatant, add 500 μL precipitation solution (1% CTAB; 50 mM Tris-HCl pH 7.5; 10 mM EDTA) and mix well, let stand for 2 min. Centrifuge at 12000g for 10-20min, discard the s...

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Abstract

The invention relates to a multiple PCR method for rapidly identifying four varieties of sea cucumbers and belongs to the field of molecular biology. The method adopts mitochondria CO I genes of four varieties of sea cucumbers as target DNA, and by sequence comparison, species specificity primers of the four varieties of sea cucumbers are designed respectively. The CO I genes of the sea cucumbersare extracted, multiple-PCR detection is carried out, thus judging the varieties of the sea cucumbers according to the molecular weight of PCR products. After the multiple-PCR detection is carried out on apostichopus japonicus, thelenota ananas, California apostichopus japonicus and Iceland sea cucumbers, the molecular weights of characteristic electrophoretic bands are 202bp, 301bp, 261bp and 358bp sequentially. The identification method has simple operation, low cost, short inspection period, good specificity and repeatability and high sensitivity, overcomes the defects of large workload, long consumed time, high cost and the like of the traditional sea cucumber morphology identification method and can realize rapid and accurate identification of the varieties of the sea cucumbers.

Description

technical field [0001] The invention relates to a method for identifying sea cucumber varieties in molecular biology, in particular to a multiplex PCR method for rapidly identifying four sea cucumber varieties. Background technique [0002] Sea cucumber is the general term for the animals of Echinodermata (Echinodermata) Holothrioider (Holothrioider). There are about 900 species of sea cucumbers in the world, and there are about 140 species in my country. There are about 40 species of sea cucumbers available for consumption worldwide. In recent years, with the continuous deepening of research on the physiology and biochemistry of sea cucumbers, the separation and identification of sea cucumber bioactive substances and their biomedical effects using modern science and technology at home and abroad, it has been found that sea cucumbers contain many important biological and pharmacological activities. substances, which also makes sea cucumber products get more and more attenti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 唐庆娟律迎春左涛段高飞李兆杰薛勇薛长湖
Owner OCEAN UNIV OF CHINA
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