Randomly amplified polymorphic dna method for quickly distinguishing the origin of sea cucumber and primers for realizing the method
A sea cucumber imitation and source technology, applied in the field of molecular biology, can solve problems such as complicated operation, high cost, and difficulty in laboratory development, and achieve the effect of overcoming high cost, low cost, and high sensitivity
Active Publication Date: 2019-03-12
CHINA NAT INST OF STANDARDIZATION
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Problems solved by technology
However, this method can only detect the species of sea cucumber, but cannot distinguish the source of sea cucumbers of the same species
[0006] As for the research on the identification of sea cucumber origin, modern technology uses near-infrared spectroscopy and ICP-MS for research, but the above-mentioned methods are characterized by complex operation, high cost, and difficult to carry out in general laboratories, so it is urgent to invent a simple and easy method. and accurate identification method
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Embodiment 1
[0033] (1) Pretreatment and species confirmation of sea cucumber samples
[0034] Purchase 9 imitation sea cucumbers from Dalian, 9 sea cucumbers from Yantai, and 8 sea cucumbers from Weihai, a total of 26, numbered sequentially, soak in Milli-Q ultrapure water for 5 hours, and set aside.
[0035] Take 30 mg of the muscle column respectively, and carry out PCR reaction with sea cucumber species-specific primers under the following reaction conditions and reaction system:
[0036]
[0037] Pre-denaturation at 95°C for 30s; denaturation at 95°C for 30s; annealing at 36°C for 30s; extension at 72°C for 1min; extension at 72°C for 4min. Do 40 cycles.
[0038] The obtained sequence was identified as A. japonicus after alignment by NCBI Blast.
[0039] Sea cucumber species-specific primers:
[0040] V3F: 5'-CGCCTGTTTATCAAAAACAT-3'
[0041] V3R: 5'-CTCCGGTTTGAACTCAGATCA-3'
[0042] (2) Extract DNA template
[0043] Weigh 80 mg of sea cucumber muscle columns for each sample, ...
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The invention relates to a method for rapidly identifying and distinguishing apostichopus japonicus from three different areas through a random amplified polymorphic DNA technology. The invention provides a primer for realizing the method, and a kit comprising the primer, wherein the primer can carry out RAPD amplification on apostichopus japonicus. The invention also relates to a random amplified polymorphic DNA method for rapidly detecting the origin regions of apostichopus japonicus; according to the method, the DNA template of a sample to be detected is extracted, then the primer D20 represented by the SEQ No.1 is taken as the random primer to carry out PCR reactions, and finally the PCR reaction product is detected by electrophoresis. Through the RAPD technology, the origin region of apostichopus japonicus can be detected rapidly, and the shortages of conventional technology such as high cost, long detection, and the like are overcome. Only through once PCR reaction and once agarose gel electrophoresis, apostichopus japonicus from Dalian, Weihai, and Yantai can be distinguished, and the method has the advantages of low cost and high sensitivity.
Description
【Technical field】 [0001] The invention belongs to the field of molecular biology, and relates to molecular biological identification of sea cucumbers, in particular to a method for quickly identifying sea cucumbers from three different origins by randomly amplifying polymorphic DNA. 【Background technique】 [0002] Sea cucumber is the collective name of sea cucumbers in the phylum Echinodermata. It is a rare seafood and has important medicinal value. Among them, Apostichopus japonicus, commonly known as Liaoshen, belongs to the order Apostichopus, Apostichopaceae, and the genus Apostichopus, and is the species of sea cucumber with the highest economic value in my country. The population is mainly distributed in shallow sea areas along the northern coasts of Liao, Hebei, Shandong, and Jiangsu, and also grows in Hokkaido, Japan, Vladivostok, Russia, and the Korean Peninsula. However, commercial interests have also led to illegal activities such as adulteration and counterfeiti...
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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 云振宇孙昭刘文刘贞张瑶孙志宏徐海燕
Owner CHINA NAT INST OF STANDARDIZATION