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PCR amplimer for environmental DNA detection of Chinese sturgeon, detection method using PCR amplimer and application of PCR amplimer

A technology for amplification primers and detection methods, which is applied in the field of environmental DNA detection of Chinese sturgeon PCR amplification primers and its detection, can solve the problems of low recognition rate, difficulty, and individual operation difficulties of acoustic detection target species, and achieve clear amplification products Stable, short amplified fragments, and high sensitivity

Active Publication Date: 2016-10-26
WATER ENG ECOLOGICAL INST CHINESE ACAD OF SCI
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AI Technical Summary

Problems solved by technology

Chinese sturgeon population monitoring and species identification are of great significance to the protection of Chinese sturgeon species. However, due to the current scarcity of natural populations of Chinese sturgeon, traditional survey methods face difficulties. Acoustic detection has a low recognition rate of target species and large errors. The large damage to Chinese sturgeon individuals has been completely banned at present; at the same time, artificial breeding and release is an important measure for the protection of Chinese sturgeon species. DNA identification of Chinese sturgeon species in artificial breeding is an important method to ensure germplasm, and clipping fin rays to obtain DNA It will cause damage to Chinese sturgeon and it is difficult to operate for larger individuals

Method used

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  • PCR amplimer for environmental DNA detection of Chinese sturgeon, detection method using PCR amplimer and application of PCR amplimer
  • PCR amplimer for environmental DNA detection of Chinese sturgeon, detection method using PCR amplimer and application of PCR amplimer
  • PCR amplimer for environmental DNA detection of Chinese sturgeon, detection method using PCR amplimer and application of PCR amplimer

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Experimental program
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Effect test

Embodiment 1

[0020] The detection method for Chinese sturgeon environmental DNA detection and identification of Chinese sturgeon species comprises the following steps:

[0021] (1) Use a 2L brand-new sterile sealed jar to collect 2L water samples on the surface of the water body to be tested, vacuum filter them with a 0.45μL microporous filter membrane, and complete the water sample suction filtration within 8 hours at room temperature. Store at ℃ until DNA extraction;

[0022] (2) Extract filter membrane DNA with Mobio Powerwater DNA extraction kit;

[0023] (3) Using water sample DNA as a template, ASCB1F (sequence: 5'-ACAATGCCACCCTTAC-3') and ASCB1R (sequence: 5'-TGTCTGCGTCTGAGTTT-3') as amplification primers, perform PCR amplification, 40μL PCR reaction system Including: upstream and downstream primers 0.5 μM each, dNTP 0.2 mM, 2U Taq DNA polymerase and 8 μL template DNA; PCR reaction conditions are as follows: 94°C pre-denaturation for 5 min; 94°C denaturation for 1 min, 56°C anneali...

Embodiment 2

[0026] Discrimination of closely related species by environmental DNA detection of Chinese sturgeon:

[0027] (1) Use the NCBI online primer search software Primer Blast to search the GenBank nucleic acid sequence database of all biological species for the amplification primers ASCB1F and ASCB1R. The search results showed that, except for Chinese sturgeon, the amplification primers only had homologous sequences in Acipenser dabryii, Acipenser dabryii, Acipenser schrenckii, Siberian sturgeon, Russian sturgeon and European huso, among which the most similar DNA sequence to Chinese sturgeon was Dabry's sturgeon, the two have a mismatch base in the core sequence of the molecular marker, which can distinguish the species of Chinese sturgeon.

[0028] (2) Three samples of caudal fin rays of Acipenser dabryii and Acipenser sinensis were taken, and tissue DNA was extracted by high-salt method as a template for PCR amplification, amplified with ASCB1 primer pair, and the amplified prod...

Embodiment 3

[0030] Effectiveness detection of PCR amplification primers of the present invention:

[0031] (1) Collect water samples of Chinese sturgeon in the artificial recirculation culture system and extract environmental DNA. The average total DNA concentration is 15 ng / μL. The environmental DNA of Chinese sturgeon can be detected with the ASCB1 primer pair. The DNA stock solution is diluted 1000 times, and then used The ASCB1 primer pair is amplified, and a clear target band can still be obtained (PCR amplification electrophoresis as shown in figure 1Shown, where the 1st and 2nd lanes are blank controls, and the 3rd to 6th lanes are the DNA detection results of aquaculture water samples).

[0032] (2) Extract the tissue DNA of Chinese sturgeon and dilute to a gradient concentration of 5×10 -1 ng / μL, 5×10 -3 ng / μL and 5×10 - 5 ng / μL, using the ASCB1 primer pair to amplify, the target bands can be obtained, indicating that ASCB1 can still detect the target DNA in a low-concentrati...

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Abstract

The invention specifically relates to a PCR amplimer for environmental DNA detection of Chinese sturgeon, a detection method using the PCR amplimer and application of the PCR amplimer, belonging to the field of biotechnology. The sequences of ASCB1F and ASCB1R of the PCR amplimer are 5'-ACAATGCCACCCTTAC-3' and 5'-TGTCTGCGTCTGAGTTT-3', respectively. The site of a DNA molecular marker of a Chinese sturgeon species is located in the mitochondrial CYTB gene of the Chinese sturgeon; a DNA fragment has a length of 138 bp and a sequence as shown in SEQ ID No. 1; the PCR amplimer is used for environmental DNA amplification, an amplification product is compared with the sequence of the site of the DNA molecular marker, and if the non-primer zones of the amplification product and the site sequence are 100% matched, a detected species is the Chinese sturgeon species. According to the invention, identification of the Chinese sturgeon species is realized by using environmental DNA detection, and a sample from a fish body is not needed, so damage to the fish body is avoided.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PCR amplification primer for detecting Chinese sturgeon environmental DNA, a detection method and an application thereof. Background technique [0002] The Chinese sturgeon (Acipenser sinensis), belonging to the order Acipenseriformes, the family Acipenseridae, and the genus Acipenser, is a typical anadromous fish. In the shallow sea area of ​​the Yangtze River estuary, the young Chinese sturgeons hatched in the upper reaches of the Yangtze River migrate down the river to the Yangtze River estuary, and enter the ocean to grow after fattening. During the breeding period from September to November every year, the mature Chinese sturgeons go upstream to the Yangtze River estuary. Spawns in the upper reaches of the Jinsha River and the lower reaches of the river. Since the closure of the Gezhouba Water Control Project in 1981, the Yangtze River Chinese sturgeon has been bl...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐念常剑波
Owner WATER ENG ECOLOGICAL INST CHINESE ACAD OF SCI
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