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Method for detecting diversity of fish species on basis of environmental DNA technology

A technology of technical detection and diversity, applied in the fields of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems such as the accuracy of the results to be improved, the detection accuracy is not enough, etc., to achieve simple sampling, accurate detection results, and convenient detection. Effect

Pending Publication Date: 2019-05-31
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Abstract
  • Description
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Problems solved by technology

[0004] There are still some defects in the above identification methods, such as insufficient detection accuracy, and the accuracy of the results needs to be improved

Method used

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  • Method for detecting diversity of fish species on basis of environmental DNA technology
  • Method for detecting diversity of fish species on basis of environmental DNA technology
  • Method for detecting diversity of fish species on basis of environmental DNA technology

Examples

Experimental program
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Effect test

Embodiment 1

[0031] A method for detecting fish species diversity based on environmental DNA technology, comprising the steps of:

[0032] 1. Water sample collection and DNA extraction

[0033] 5 L of surface water samples were collected from 9 sampling points in the waters of the Fuchun River, brought back to the laboratory and placed in a 4°C freezer, and the water samples were filtered within 24 hours, using a filter membrane with a pore size of 0.45 μm. The eDNA in the filter membrane was extracted using the kit EZNA waterDNA kit produced by Omega Company. The extracted DNA was dissolved in TE buffer and stored at -20°C for future use.

[0034] 2. PCR amplification and quality inspection

[0035] Cytb and 16S primers were used to amplify water sample DNA, and the primer sequences are shown in Table 1. The 25 μL reaction system included: 2.5 μL 10×buffer, 2 μL dNTP (2.5 mmol·L -1 ), each 1 μL of forward and reverse primers (10 μmol﹐L -1 ), 0.15μL Taq enzyme (5 UoμL -1 ), 1 μL DNA t...

Embodiment 2

[0052] The same sampling point was selected twice in the Qiandao Lake National Aquatic Germplasm Resources Protection Area to collect 5L of surface water samples, and they were brought back to the laboratory and placed in a 4°C freezer. DNA extraction, PCR amplification and quality inspection, target fragment library construction, sequencing and data processing, fish taxonomic annotation methods are the same as in Example 1. The results are shown in Table 4. A total of 9 fish species were annotated with Cytb F / Cytb R primers, and 27 fish species were annotated with 16S F / 16S R primers. Most of the annotated species are species that can be collected in Qiandao Lake by traditional fishing methods. In addition to the common indigenous fishes detected in Qiandao Lake, some exotic species and hybrid species were also detected, such as tilapia, fork-tailed betta, round-tailed betta, spoon-nose sturgeon, Russian sturgeon, hybrid sturgeon, bream×triangle bream.

[0053]

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Abstract

The invention discloses a method for detecting diversity of fish species on the basis of an environmental DNA technology. The method comprises the following steps of (1) conducting sampling; (2) extracting eDNA; (3) performing PCR amplification; (4) recovering and purifying target fragments and constructing a target fragment library; (5) conducting OTU clustering; (6) creating a fish comparison database; (7) conducting comparison analysis on representative sequences of OTUs with data information of the created fish database to determine the species diversity composition of fishes in a to-be-detected water area. According to the method, there is no need to catch the fishes, sampling is simple, and the fish bodies cannot be damaged; detection is convenient, and there is no need to conduct fish appearance identification; detection is simple and fast, the precision is high, and a detection result is accurate.

Description

technical field [0001] The invention belongs to the field of molecular ecology, and in particular relates to a method for detecting fish species diversity based on environmental DNA technology. Background technique [0002] Environmental DNA (environmental DNA, eDNA) refers to free DNA molecules released from organisms. eDNA technology refers to the method of qualitative or quantitative analysis by sequencing technology after extracting DNA fragments from environmental samples. [0003] Traditional species detection mainly uses on-site surveys and morphological methods for species identification. It is better for species that are easy to sample and have large individuals. However, due to the particularity of the water environment and the fact that fish flow in the water It is highly destructive and difficult to catch. Traditional species detection methods have poor effects on it and cause harm to fish. Therefore, the use of eDNA technology to analyze the species diversity ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869
Inventor 张爱菊周志明刘金殿王俊郝雅宾于喆
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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