阅读说明：本技术 一种利用环境沉积物样品监测淡水底栖动物的方法 (Method for monitoring freshwater benthonic animals by utilizing environmental sediment samples ) 是由 金珂 杨江华 张效伟 于 2020-06-11 设计创作，主要内容包括：本发明属于底栖动物鉴定技术领域,公开了一种利用环境沉积物样品监测淡水底栖动物的方法,包括以下步骤：1)首先采集环境沉积物样本,清洗后保存；2)向步骤1)保存的环境沉积物样本中加入无水乙醇进行浸提处理,处理一段时间后取混合均匀的浸提液,将所述浸出液进行真空离心,弃上清液,获得干燥的组织残渣,进行DNA提取；3)利用提取得到的DNA条形码片段进行扩增,得到扩增产物；4)将扩增产物进行测序与分析。本发明的方法将样品首先采用无水乙醇浸提,使底栖动物DNA均匀释放到溶液中,达到均质的状态,通过采集含有DNA样品的乙醇溶液,离心浓缩后进行DNA提取,可实现大范围环境样品的准确监测。(The invention belongs to the technical field of benthonic animal identification, and discloses a method for monitoring freshwater benthonic animals by using an environmental sediment sample, which comprises the following steps: 1) firstly, collecting an environmental sediment sample, cleaning and then storing; 2) adding absolute ethyl alcohol into the environmental sediment sample stored in the step 1) for leaching treatment, taking the uniformly mixed leaching liquor after a period of treatment, carrying out vacuum centrifugation on the leaching liquor, discarding supernatant fluid to obtain dry tissue residues, and carrying out DNA extraction; 3) amplifying the DNA barcode fragment obtained by extraction to obtain an amplification product; 4) sequencing and analyzing the amplified product. According to the method, the sample is firstly extracted by absolute ethyl alcohol, so that DNA of the benthonic animals is uniformly released into the solution to reach a homogeneous state, and accurate monitoring of a large-range environmental sample can be realized by collecting the ethanol solution containing the DNA sample, carrying out DNA extraction after centrifugal concentration.)

1. A method of monitoring freshwater benthonic animals using environmental sediment samples, comprising: the method comprises the following steps:

1) firstly, collecting an environmental sediment sample, cleaning and then storing;

2) adding absolute ethyl alcohol into the environmental sediment sample stored in the step 1) for leaching treatment, taking the uniformly mixed leaching liquor after a period of treatment, carrying out vacuum centrifugation on the leaching liquor, discarding supernatant fluid to obtain dry tissue residues, and carrying out DNA extraction;

3) amplifying the DNA barcode fragment obtained by extraction to obtain an amplification product;

4) sequencing and analyzing the amplification product obtained in the step 3).

2. The method of monitoring freshwater benthic animals using environmental sediment samples according to claim 1, wherein: and in the step 1), the cleaned sample is frozen and stored at the temperature of-20 to-80 ℃ for 1 to 3 days.

3. The method for monitoring freshwater benthic animals according to claim 1 or 2, wherein the environmental sediment sample is selected from the group consisting of: the time of leaching treatment in the step 2) is 3-30 days.

4. The method of monitoring freshwater benthic animals according to claim 3, wherein the environmental sediment sample is selected from the group consisting of: in the step 2), 3-5 times of volume of absolute ethyl alcohol is added into the sediment sample in a conversion mode of converting 1g of sediment into 1 ml.

5. The method of monitoring freshwater benthic animals according to claim 4, wherein the environmental sediment sample is selected from the group consisting of: the vacuum centrifugation conditions were: centrifuging at 300-1000 rpm for 2-3 hours.

6. The method of monitoring freshwater benthic animals according to claim 5, wherein the environmental sediment sample is selected from the group consisting of: and 2) using an OMEGA Tissue kit to extract DNA from the Tissue residues.

7. The method of monitoring freshwater benthic animals according to claim 4, wherein the environmental sediment sample is selected from the group consisting of: the primer sequence adopted in the amplification in the step 3) is as follows:

an upstream primer: mlCOIintF: GGWACWGGWTGAACWGTWTAYCCYCC, respectively;

a downstream primer: jgHCO 2198: TAIACYTCIGGRTGICCRAARAAYCA are provided.

8. The method for monitoring freshwater benthic animals according to claim 6 or 7, wherein the environmental sediment sample is selected from the group consisting of: the PCR amplification conditions are as follows:

s1, constructing a PCR amplification system: 25 ul PrimeStarMax amplification enzyme, 5 ul DNA template, total amount of about 20ng, 1.25 ul each of upstream and downstream primers, 17.5 ul enzyme-free water;

s2, pre-denaturation at 98 ℃ for 3 minutes;

s3, denaturation at 98 ℃ for 15 seconds, renaturation at 46-48 ℃ for 30 seconds, extension at 72 ℃ for 45 seconds, and repeating for 35 cycles;

s4, and extension is carried out at 72 ℃ for 5 minutes to obtain an amplification product.

9. The method for monitoring freshwater benthonic animals using environmental sediment samples of claim 8, wherein: the step 4) comprises the following steps:

4-1), sequencing the amplification product by high-throughput sequencing technology;

4-2), processing the sequencing result by using software, and performing species annotation by contrasting an NCBI database;

4-3), and manually screening the species annotation result to serve as a biological monitoring result.

10. The method of monitoring freshwater benthic animals according to claim 9, wherein the environmental sediment sample comprises: in the step 4-2), the software for processing the sequencing result comprises qiime and usearch.

Technical Field

The invention belongs to the technical field of benthonic animal identification, and particularly relates to a method for monitoring freshwater benthonic animals by using an environmental sediment sample.

Background

The large-scale benthonic animals in fresh water are one of key monitoring objects for evaluating the ecological health of the drainage basin, the current monitoring process mainly comprises the steps of collecting sediment samples, manually picking and morphologically identifying, and the process has the limitations of long time consumption, high labor cost, high identification level required by experts and the like. By extracting DNA in a sediment sample and tracing the type of the sediment in a PCR amplification mode, the defects of long time consumption, high labor cost and complex operation in the prior art by utilizing morphological identification can be effectively overcome.

Through retrieval, a relevant application is disclosed in the prior art aiming at the biological DNA extraction mode in a sediment sample, for example, the application with Chinese patent application number of 200510079754.1 and the publication date of 2008, 12 and 3 discloses a method for simultaneously extracting microbial metagenome DNA and total RNA from marine sediments.

Further, as in the application with the Chinese patent application No. CN200610136833.6 and the publication date of 2009-06-10, the method for extracting the total genome of deep sea sediments includes the treatment of samples and the extraction of DNA. Dissolving a centrifugally collected deep sea sediment sample by using basic salt of sterile artificial seawater, adding a high-concentration cell lysate into an experimental sample, lysing cells in a boiling water bath, adding a phenol/chloroform/isoamylol mixed solution, centrifuging, repeatedly extracting, and collecting a supernatant; precipitating the supernatant with anhydrous ethanol, centrifuging, and collecting precipitate; washing the precipitate with ethanol, vacuum drying the precipitate, and dissolving the precipitate to obtain the genome DNA extracting solution. The method of the application applies the principles of microbiology, biochemistry, molecular biology, physics and chemistry to solve the problems of low efficiency, poor universality, low yield, high cost, large sample consumption and the like in the process of extracting the total genomic DNA of deep-sea microorganisms specific to a habitat, thereby improving the yield and the variety quantity of the DNA in a deep-sea sample and leading the molecular ecology research result to be more comprehensive and more accurate.

The COI gene is a coding gene of cytochrome oxidase subunit I in animal cell mitochondria, has a sequence with large difference among species, and is one of the common species identification barcode regions. Through retrieval, the Chinese patent application No. 201910840869.X, published as 2020.03.20, discloses a high-throughput sequencing method for a benthonic animal COI gene and application thereof, and the method of the application mainly comprises the following steps: s1, taking a DNA sample of the benthonic animal mixed sample as a template, and carrying out PCR amplification on the COI gene; s2, carrying out PCR amplification by taking the PCR amplification product in the step S1 as a template, wherein an amplified primer pair comprises a forward primer and a reverse primer, the primer pair comprises a pyrosequencing high-throughput sequencing joint, each sample corresponds to one pyrosequencing high-throughput sequencing joint, and a PCR product is obtained and is used as a DNA library; and S3, performing quality detection on the DNA library obtained in the step S2, and then selecting a qualified DNA library for empCR amplification and high-throughput sequencing. The sequencing method of the application has high flux, can obtain COI standard barcode gene-658 bp data by one-time sequencing, has high accuracy and provides larger taxonomic resolution. For benthonic animal samples, the collection and processing method disclosed in the examples is as follows: firstly, collecting benthonic animal samples in different habitats by utilizing a Soppe net or a Peterson mud sampler, collecting 3 parallel samples at each point, cleaning, roughly picking, putting into a 300mL standard sample bottle, and fixedly storing by 95% alcohol. The biological mixed sample prepared above was subjected to DNA extraction using a tissue genome DNA extraction kit, and the DNA concentration was measured and diluted in a lump to a final concentration of 5 ng/. mu.l.

Based on the defects of the prior art, the method for monitoring the freshwater benthonic animals in the environmental sediment sample, which can improve the monitoring accuracy and has a wide coverage range, needs to be invented.

Disclosure of Invention

1. Problems to be solved

Drawings

FIG. 1 is a gel map of detection of a COI gene library in example 1, wherein FIGS. 1A to 1D respectively show DNA gel maps extracted from different collection sites;

FIG. 2 shows the results of annotation of various groups in example 1;

FIG. 3 is a graph showing the ratio of the number of groups OUT in example 1;

FIG. 4 is a comparison of species ratios of various groups of morphological and eDNA assays of example 1;

FIG. 5 is a comparison of the results of the quantitative relationship fit between the morphological examination and the eDNA examination in example 1; fig. 5A shows that there is a significant positive correlation between the log values of the relative abundance of biomass and the log values of the relative abundance of the sequence number in the families of wormcast and chironomidae, respectively, and fig. 5B shows that there is a significant positive correlation between the log values of the relative abundance of biomass and the log values of the relative abundance of the sequence number in the genus of periploris;

FIG. 6 shows a comparison of DNA concentrations obtained by the three methods of comparative example A, comparative example B and example 1;

FIG. 7 is a comparison of the sequence sources obtained by the method of example 1 and the two methods of comparative example A.

Detailed Description

The invention is further described with reference to specific examples.