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COI gene based PCR-RFLP discrimination method of seventeen economic sea cucumbers of Stichopodidae

A technology of PCR-RFLP and sea cucumber, applied in biochemical equipment and methods, microbe measurement/inspection, etc., can solve the problems of lack of systematic research and few applications, and achieve accurate detection, low requirements for instruments and equipment, and wide application range wide effect

Active Publication Date: 2014-07-16
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Mitochondrial DNA COI sequence is widely used in the research of sea cucumber phylogenetic evolution and species identification. COIPCR-RFLP is a simple and fast method for species identification. At present, this method is rarely used in sea cucumber species identification and lacks systematic research.

Method used

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  • COI gene based PCR-RFLP discrimination method of seventeen economic sea cucumbers of Stichopodidae
  • COI gene based PCR-RFLP discrimination method of seventeen economic sea cucumbers of Stichopodidae
  • COI gene based PCR-RFLP discrimination method of seventeen economic sea cucumbers of Stichopodidae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: PCR-RFLP identification of Apostichopus japonicus samples

[0017] a. PCR amplification of the COI fragment of the Apostichopus japonicus sample:

[0018] The DNA of the Apostichopus japonicus samples was extracted by conventional methods, and then the COI fragment was amplified by PCR using it as a template. The PCR amplification reaction volume was 50 μL, and 200 μL PCR tubes were placed in an ice bath, followed by adding 37.6 μL sterile water, 5 μL 10 × PCRBuffer (including 15mMMg 2+ ), 1μL 10uMCOIe-F primer, 1μL 10uMCOIe-R primer, 4μL 2.5mM dNT Pmixture, 0.4μL 5U / μL TaKaRaTaqTM enzyme, 1μL Apostichopus japonicus sample DNA template; ABIVeriti 96-well thermal cycler was used for amplification, and the reaction conditions were pre-denaturation at 94°C for 2 minutes , followed by 35 cycles of denaturation at 94°C for 30 seconds, renaturation at 48°C for 30 seconds, and extension at 72°C for 45 seconds, with a final extension at 72°C for 7 minutes. The COIe...

Embodiment 2

[0028] Example 2: Identification of Apostichopus japonicus samples by PCR-RFLP

[0029] Steps a-c are the same as those in Example 1, except that Apostichopus japonicus is used as a sample.

[0030] d. Judgment of test results

[0031] After electrophoresis, take pictures under the gel imaging system (Bio-Rad Company) to obtain the restriction electrophoresis diagrams of the two groups of endonucleases DdeI / SfcI and BstNI / Sau3AI in the COI fragment of the sea cucumber sample (see figure 1); the added 50bp DNALadder was set as the molecular weight standard, and the Quantityone software estimated that: ① The estimated value of the four bands cut by DdeI / SfcI double enzyme was 254.18bp, 194.10bp, 124.82bp and 75.26bp; ②BstNI / Sau3AI double enzyme cut The estimated values ​​of the three bands are 316.33bp, 212.04bp and 161.56bp. Compared with Table 1, the number of bands is the same as that of A. japonicus, and the estimated value of each electrophoresis band matches the correspo...

Embodiment 3

[0032] Example 3: Identification of Apostichopus japonicus samples by PCR-RFLP

[0033] Steps a-c are the same as in Example 1, but Apostichopus japonicus is used as a sample.

[0034] d. Judgment of test results

[0035] After electrophoresis, take pictures under the gel imaging system (Bio-Rad Company) to obtain the restriction electrophoresis images of two groups of endonucleases DdeI / SfcI and BstNI / Sau3AI in the COI fragment of A. japonicus samples (see figure 1 ); the added 50bp DNALadder was set as the molecular weight standard, and Quantityone software estimated that: ① The estimated values ​​of the three bands cut by DdeI / SfcI double enzymes were 254.18bp, 194.10bp and 123.25bp; ② The three bands cut by BstNI / Sau3AI double enzymes Band estimates are 319.39bp, 206.48bp and 160.07bp. Compared with Table 1, the number of bands is the same as that of A. japonicus, and the estimated value of each electrophoresis band matches the corresponding fragment size in the standard...

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Abstract

The invention discloses a COI gene based PCR-RFLP discrimination method of seventeen economic sea cucumbers of Stichopodidae. The method comprises the following steps: 1, extracting DNA of a Stichopodidae sea cucumber to be detected as a detected sample, using the DNA as a template, and using primers COIe-F:5'-ATAATGATAGGAGGRTTTGG-3', COIe-R:5-GCTCGTGTRTCTACRTCCAT-3'PCR' to amplify a CO I fragment, wherein A is A / G; and 2, carrying out enzyme digestion on the CO I fragment by using a restrictive endonuclease group DdeI / SfcI or BstNI / Sau3AI, carrying out electrophoresis on the above obtained enzyme digestion product, estimating the size of each of the electrophoretic bands of samples to be detected, contrasting the number and the size of the electrophoretic bands in an electrophoretogram and the estimated value of the size with the reference band spectra of the seventeen sea cucumbers, and finding the standard spectral bands coupled with the number and the size of the electrophoretic bands, wherein the types represented by the standard spectral bands are the type of the detected sample. The method has the advantages of wide application range, simple operation, and accurate and fast detection, is especially suitable for the identification of a large batch of commodity sea cucumbers, so the method is a scientific method for identifying the sea cucumbers of Stichopodidae, and provides technologic supports for the standardization of the commodity market of the sea cucumbers and the protection of the sea cucumber resources.

Description

Technical field: [0001] The invention relates to a simple molecular identification method of sea cucumber species, in particular to a PCR-RFLP identification method based on COI gene of 17 economic sea cucumbers of Apostichopus japonicus. Background technique: [0002] Sea cucumber has high nutritional value and medicinal value, and is considered to be a traditional delicacy and tonic in my country, ranking one of the "Eight Treasures". There are more than 1,400 species of sea cucumbers recorded worldwide, and there are about 50 species of sea cucumbers that can be eaten, mainly from the families Holothuriidae and Stichopodidae. Compared with the species of sea cucumbers, sea cucumbers of the family Apostichopus have larger verrucous feet on the back, and the bone fragments are mostly table-shaped, without buttons, but with C-shaped bodies or double-branched rods, or simple granules. As a valuable seafood product, sea cucumber is usually processed as dry or frozen products ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/683C12Q1/686C12Q2531/113C12Q2521/301
Inventor 夏建军胡超群任春华罗鹏于宗赫
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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