Species-specific PCR method for rapidly identifying ten sea cucumber species

A kind of specific and specific technology, applied in the field of molecular biology, can solve the problem of high cost, and achieve the effect of high specificity and low cost

Inactive Publication Date: 2014-01-01
ZHANJIANG NORMAL UNIV
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  • Claims
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Problems solved by technology

Real-time quantitative PCR metho

Method used

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  • Species-specific PCR method for rapidly identifying ten sea cucumber species
  • Species-specific PCR method for rapidly identifying ten sea cucumber species

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Embodiment

[0022] Identification of Dried Sea Cucumber Species

[0023] (1) Extraction of DNA template: ① Cut about 10 mg of sea cucumber muscle, rinse with distilled water, and blot dry with filter paper. ② Use the Marine Animal Genomic DNA Extraction Kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract DNA as a template according to the instructions, and store it at -20 °C for later use.

[0024] (2) Species-specific PCR amplification of specific fragments of the 16S rRNA gene: ① The total reaction system is 50 μl, including: 38.5 μl water; 5 μl 10 × Ex Taq buffer (containing 20 mM Mg 2+ ); 1 μl dNTP (10 mM); 2 μl forward primer 16Sar (10 μM); 2 μl reverse primer (10 μM); 1 μl DNA template; 0.5 μl Ex Taq DNA polymerase; forward primer 16Sar used : 5'-CGCCTGTTTATCAAAAACAT-3'; species-specific reverse primers were: Actinopyga mauritiana Reverse primer 16Smau-t: 5'-CGAGTTAGGTTAGAAAATTTCC-3', Stichopus naso Reverse primer 16Snas-t: 5'-GGGTATACTTAGTTTTTCAGG-3', Cucum...

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Abstract

The invention relates to a species-specific PCR method for rapidly identifying ten sea cucumber species, and belongs to the molecular biology field. The method comprises the following steps: respectively designing species-specific primers of the ten species of sea cucumber through sequence alignment by treating mitochondrial 16SrRNA gene of the ten species of sea cucumber as target gene, extracting sea cucumber mitochondrial gene, carrying out specific PCR amplification detection, determining the sea cucumber species comprising Actinopygamauritiana, Stichopusnaso, Cucumariafrondosa, Holothuriaedulis, Holothuriaatra, Holothuriaflavomaculata, Holothuriamammata, Holothuriabacilla, Holothurianotabilis and Holothuriamoebii according to the existence or inexistence of a PCR amplification band, carrying out specific PCR amplification to respectively obtain 274bp, 276bp, 264bp, 275bp, 270bp, 273bp, 274bp, 276bp, 276bp and 270bp specific fragments.

Description

technical field [0001] The invention relates to a method for identifying sea cucumber varieties in molecular biology, in particular to a species-specific PCR method for rapidly identifying 10 sea cucumber varieties, and belongs to the technical field of molecular biology. Background technique [0002] Sea cucumber, belonging to Holothurioidea, is a marine mollusk that lives on the seashore to 8,000 meters. It has a history of more than 600 million years. Sea cucumbers feed on seabed algae and plankton. Sea cucumbers are covered with thorns and are widely distributed in oceans all over the world. There are many kinds of sea cucumbers along the coast of the South my country Sea. There are more than 20 kinds of sea cucumbers available for consumption. Sea cucumbers are as famous as ginseng, bird's nest and shark's fin, and are one of the eight treasures in the world. Sea cucumber is not only a precious food, but also a valuable medicinal material. According to the records in ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6888
Inventor 文菁曾玲
Owner ZHANJIANG NORMAL UNIV
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