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PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species

A PCR-RFLP, sea cucumber technology, applied in the field of identifying sea cucumber species, can solve the problems of low throughput, long time, heavy workload, etc., and achieve the effects of good specificity, short typing time, and easy operation.

Inactive Publication Date: 2013-04-03
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, morphological identification has its limitations: 1) phenotypic plasticity and genetic variability can easily lead to incorrect identification; 2) morphological methods cannot identify cryptic taxa that are prevalent in many populations; 3) morphological identification is limited by Restrictions on biological sex and developmental stage affect the accuracy of identification; 4) The workload is heavy and time-consuming
However, these two methods need to be sequenced and compared with online data to draw a phylogenetic tree to draw conclusions, which takes a long time, high cost, and low throughput

Method used

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  • PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species
  • PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species
  • PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Identification of known sea cucumber species

[0037] The extracted sea cucumber DNA was amplified by PCR, and the amplified product was subjected to restriction endonuclease digestion experiment. The total PCR-RFLP reaction system was 30 μL, including: 2 μL of 10× enzyme digestion buffer, 10 μL of PCR product, and the corresponding specific Concentration of specific endonuclease: 1FDU / μL0.5μL (for example, the specific endonuclease corresponding to California sea cucumber: SSPⅠ; the specific endonuclease corresponding to North Atlantic sea cucumber: EcoRⅤ; the specific endonuclease corresponding to sea cucumber : SSPⅠ; the specific endonuclease corresponding to plum blossom ginseng: EcoRⅤ; the specific endonuclease corresponding to Haitian melon: ScaⅠ; the specific endonuclease corresponding to American ginseng: PvuⅡ; the specific endonuclease corresponding to boot ginseng: EcoRⅤ, SSPⅠ), the rest were made up with water, 37°C for 30min, and the digested products were d...

Embodiment 2

[0040] Identification of Unknown Sea Cucumber Species

[0041] The extracted sea cucumber DNA was amplified by PCR, and the amplified product was subjected to a restriction enzyme digestion experiment. The total reaction system was 30 μL, including: 2 μL of 10× enzyme digestion buffer, 10 μL of PCR product, and the concentration of each of the four enzymes Add 0.5 μL to each 1FDU / μL, 16 μL water, 30 min at 37°C, and detect the digested product by polyacrylamide gel electrophoresis. According to the enzyme digestion results, it was judged that the unknown sample belonged to California japonicus, North Atlantic melon ginseng, imitation japonicus japonicus, plum flower ginseng, Haitian melon, American meat ginseng or boot ginseng. For example, if two bands with a size of 190bp and 498bp are obtained, the sea cucumber to be identified is Apostichopus californica, and the others are similar.

Embodiment 3

[0043] Identification of sea cucumbers with lost external morphological characteristics

[0044] In order to prove that the method can identify sea cucumbers that have lost their external morphological characteristics, the present invention takes sea cucumber enzymatic hydrolyzate as an example to verify. The extracted DNA from the enzymatic solution was amplified by PCR, and the amplified product was subjected to restriction endonuclease digestion experiment. The total reaction system was 30 μL, including: 2 μL of 10× digestion buffer, 10 μL of PCR product, four enzymes each 0.5 μL of each concentration of 1 FDU / μL, 16 μL of double distilled water, 37°C for 30 min, and the digested products were detected by polyacrylamide gel electrophoresis. The result is as image 3 , the electrophoretic pattern of the digested product was consistent with that of the imitated sea cucumber, that is, three bands with sizes of 190bp, 221bp, and 281bp were obtained, indicating that the enzymat...

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Abstract

The invention discloses a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species. The PCR-RFLP method includes the steps of firstly extracting DNA (deoxyribonucleic acid) modules of sea cucumbers; subjecting specificity fragment of a CO I (coenzyme I) gene to PCR amplification to obtain a fragment about 690bp; secondarily subjecting amplified product to restriction enzyme digestion; and finally subjecting the enzyme-digested product to electrophoresis detection. Different sea cucumbers obtain stripes in different numbers and sizes, and selected specificity incision enzymes respectively include: Californian paracanthurus cucumber: SSP I, North Atlantic Ocean cucumaria: EcoR V, apostichopus japonicas: SSP I, thelenota ananas: EcoR V, acaudina molpadioidea: Sca I, isostichopusbadionotus: Pvu II, and actinopyga mauritiana: EcoR V and SSP I. The PCR-RFLP method overcomes the defects that a conventional morphological identification method is prone to affecting in accuracy, is heavy in workload and long in consumption time and the like. The PCR-RFLP method can be completed by only one-time PCR reaction, one-time enzyme digestion reaction and one-time polypropylene gel electrophoresis, and is simple to operate, short in parting time and good in specificity.

Description

technical field [0001] The invention relates to a method for identifying sea cucumber species in molecular biology, in particular to a PCR-RFLP method for identifying seven sea cucumber species. Background technique [0002] Sea cucumber is a traditional marine food and medicine resource in my country. Due to the large differences in the nutritional value and economic value of different types of sea cucumbers, some unscrupulous traders often deceive consumers by using false ones and shoddy ones. Sea cucumbers that have lost their original form are more prone to counterfeiting. [0003] The traditional sea cucumber species identification method is morphological identification, which is mainly based on the external morphology and anatomical characteristics of sea cucumbers: external characteristics include the shape of tentacles, the presence or absence of warty feet and tube feet, their shape and distribution characteristics, etc.; anatomical characteristics mainly refer to ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 唐庆娟郑荣律迎春王玉明李兆杰薛勇王静凤薛长湖
Owner OCEAN UNIV OF CHINA
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