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Method for detecting diversity of fish species by using environmental DNA technology

A technology of diversity and species, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problem that species cannot be monitored, and achieve the effect of accurate detection results and various types

Pending Publication Date: 2021-09-07
邢迎春 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are still certain problems in the detection amount of this method, and more species cannot be monitored within the same detection time.

Method used

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  • Method for detecting diversity of fish species by using environmental DNA technology
  • Method for detecting diversity of fish species by using environmental DNA technology
  • Method for detecting diversity of fish species by using environmental DNA technology

Examples

Experimental program
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Effect test

Embodiment 1

[0027] The purpose of this embodiment is to provide a method for detecting fish species diversity using environmental DNA.

[0028] 1. Design primers

[0029] According to the primer design, the sequence of the vertebrate 12S rRNA universal primer is shown in Table 1, and the amplified target band is 106bp. In order to label the sample sequences of 45 sampling points and negative controls (negative control during sampling, negative control during eDNA extraction process, negative control during PCR process), the same specific label was added to the 5' ends of the forward and reverse primers. The tag is designed using OLIGOTAG software, consisting of six nucleotides, and 2 or 3 N bases are added in front of the tag, and the tag tag is shown in Table 2. The 48 sets of 12s rRNA universal primers after adding the tag were sent to Biosynthesis Co., Ltd. for synthesis, and the primer set was obtained. The aforementioned primer set was sequenced. The primer set is the adjusted 12s ...

Embodiment 2

[0045] The purpose of this example is to use the method provided in Example 1 to detect water samples.

[0046] 1. Sampling

[0047] Adopt according to the method in embodiment 1, altogether 135 samples are arranged at 45 sampling points in Poyang Lake, the sampling point information map is as follows figure 1 shown. Water samples were vacuum filtered using a 0.45 μm mixed cellulose lipid membrane (MCE: Whatman Company) within 24 h. Add a negative control with 1L mineral water as a sample at each testing point to detect whether the sample is contaminated during storage, transportation, and extraction. After each sample was filtered, the filters were placed in 2 mL centrifuge tubes and stored frozen until DNA extraction. Samples from different sampling points are kept in isolation to prevent cross-contamination between samples.

[0048] 2. Filter

[0049] According to the method shown in Example 1, the aforementioned samples were vacuum filtered, and the spare samples were...

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Abstract

The invention provides a method for detecting fish species diversity by using environmental DNA, and relates to the technical field of molecular ecology. The method for detecting fish species diversity by using the environmental DNA comprises the following steps: collecting a water sample of a place to be detected, filtering, extracting eDNA to obtain eDNA of the water sample to be detected, carrying out PCR amplification by using a primer group to obtain an amplification product, carrying out high-throughput sequencing on the amplification product to obtain sequence fragments, splicing the sequence fragments, and carrying out OTU cluster analysis on the species diversity. The primer group comprises an upstream primer and a downstream primer, the nucleotide sequence of the forward primer is as shown in SEQID.NO 1, and the nucleotide sequence of the reverse primer is as shown in SEQID.NO 2. The method has the advantages that the method can be used for detecting the diversity of fish species, especially the diversity of lake aquatic organisms, the detection result is accurate, and multiple species can be detected.

Description

technical field [0001] The invention relates to the technical field of molecular ecology, in particular to a detection of fish species diversity using environmental DNA technology. Background technique [0002] Environmental DNA (environmental DNA, eDNA) was first proposed in the late 1970s, and refers to free DNA molecules released from organisms, existing in soil, sediment, air and water and other environments. Environmental DNA extraction technology was first used to isolate and purify the DNA of microorganisms in sediments. In recent years, environmental DNA technology has been applied in ecological research fields such as biodiversity analysis, biomass estimation, rare species discovery, biological invasive species monitoring, and community phylogenetic reconstruction. Through environmental DNA technology, sequencing technology can be used to qualitatively or quantitatively analyze species types and species biomass in the environment after extracting DNA fragments. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869
CPCC12Q1/6869C12Q2531/113C12Q2535/122C12Q2537/165C12Q2545/113
Inventor 邢迎春赵亚辉高婉茹
Owner 邢迎春
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