Quantitative method for fish diversity based on eDNA and sampling and filtering device thereof

A filtration device, a variety of technologies, applied in sterilization methods, biological material sampling methods, support/immobilization methods of microorganisms, etc., can solve the problem of affecting eDNA, the accuracy of analysis results and the high experimental cost, cross-contamination and degradation, etc. problems, to achieve high specificity, meet cloning and high-throughput sequencing, and avoid cross-contamination

Active Publication Date: 2019-04-09
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of cross-contamination, degradation and other risks that eDNA still exists in samples during the experimental stage, which

Method used

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  • Quantitative method for fish diversity based on eDNA and sampling and filtering device thereof
  • Quantitative method for fish diversity based on eDNA and sampling and filtering device thereof
  • Quantitative method for fish diversity based on eDNA and sampling and filtering device thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0037] Embodiment 1: An eDNA-based quantitative method for fish diversity and its sampling and filtering device, including the following steps:

[0038] S1. Using sterilized sampling bottles, select aquatic organisms to protect the lake and its upstream and downstream reservoirs for multiple sampling to collect 1L samples. Set a sampling point every one kilometer, and set 3 parallel samples for each sampling point. The sampling interval is 6 months;

[0039] S2. Use a sterilized quantitative sampling filter device based on eDNA fish diversity to filter the collected samples, remove the microporous filter membrane after filtering, and place the microporous filter membrane in a refrigerator at -20°C Cryopreservation in

[0040] S3. Take out the microporous filter membrane and place it in a liquid nitrogen mortar to crush, pour the debris into a 2mL centrifuge tube and store it for later use;

[0041] S4. Take out the debris of the sample filter membrane, and use a DNA extraction reagen...

Example Embodiment

[0053] Example 2: Based on Example 1, but the difference is that intensive sampling was carried out at 7 sampling points upstream and downstream of Huairou Reservoir, with a sampling volume of 1L, and the collected samples used a sterilized eDNA-based fish A variety of quantitative sampling and filtering device. The filter membrane is placed in a liquid nitrogen mortar and crushed. DNA extraction experiments are performed within 24 hours. The extracted DNA samples are placed in an ultra-micro spectrophotometer for concentration detection, and the most covered species are selected. Identify the universal primers with the highest accuracy. Use primers for diversity PCR amplification. Take 2uL of purified PCR product and connect it to the vector, transform competent cells, and incubate overnight at 37°C. After cloning, pick 50 single colonies and use The primers were verified by colony PCR, and the PCR products were detected by agarose gel electrophoresis and confirmed to be the ta...

Example Embodiment

[0055] Example 3: Based on Examples 1 and 2, but the difference is that 4 sampling points with gene sequences of the broadfin snails were selected for sampling, and the collected samples were subjected to DNA extraction experiments. According to the gene sequences of the BLAST comparison results, Download the full gene sequence of Zaccoplatypus from the NCBI database, and perform primer screening in Primer5.5 software to obtain the upstream and downstream primer fragments of the target gene, and perform primer synthesis. Use the collected sample as a template and use the target gene The primers are used for PCR amplification, the recovered PCR products are subjected to TA cloning, the target colony is selected for plasmid extraction, and the extracted plasmid is used as an absolute quantitative standard. The RealTime PCR reaction system is configured according to the DNA sample, and the RealTime PCR amplification experiment is carried out for each sample. Repeat the test 3 times...

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Abstract

The invention discloses a quantitative method for fish diversity based on eDNA and a sampling and filtering device thereof, and belongs to the field of fish diversity research. According to the method, sample DNA extraction is performed by adopting a filtering method, the eDNA-based quantitative sampling and filtering device for fish diversity is adopted, and stirring is performed at the same timeof multiple suction filtration, so that an experimental period is greatly shortened, and meanwhile, samples can be effectively avoided from being cross-contaminated in the experimental stage; the filtered samples are used for DNA extraction within 24 hours, which can effectively reduce DNA degradation; by adopting human tissue and a blood extract as an extractant, the maximum extraction amount can be obtained, and the extracted DNA concentration and purity are relatively high; by adopting DNA clone sequencing, the experiment cost can be reduced; an absolute quantitative PCR method is used forquantitative research of fish, absolute quantification can be used for determining the copy number or concentration of genes in the samples by a standard curve and a dissolution curve, the specificity is high and the accuracy is good.

Description

technical field [0001] The invention relates to the field of fish diversity research, in particular to an eDNA-based quantitative method for fish diversity and a sampling and filtering device thereof. Background technique [0002] As the highest trophic level in aquatic ecosystems, fish can reflect the productivity and biodiversity levels of aquatic ecosystems. The survey methods of traditional fish survey include visit and statistics, random fishing and electric fish and other methods. However, these survey methods often take months or even years for researchers to capture, identify and classify fish in larger watersheds, and are not time-sensitive. In addition, the fishing method usually uses electric fish, but the magnitude of the current is difficult to control. In order to increase the reliability of the experiment during part of the capture process, many fish were electrocuted to death, causing significant damage to the ecosystem. In recent years, the hydroacoustic m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6851C12M1/26C12M1/12C12M1/02
CPCC12Q1/6806C12Q1/6851C12Q1/6869C12Q2531/113C12Q2535/122C12Q2563/107
Inventor 陈贺刘子方
Owner BEIJING NORMAL UNIVERSITY
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