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A Quantitative Method of Fish Diversity Based on Edna and Its Sampling and Filtering Device

A filtration device and a variety of technologies, which can be used in sterilization methods, biological material sampling methods, support/immobilization methods for microorganisms, etc., which can solve the problems of high accuracy of analysis results and high experimental costs, affecting eDNA, cross-contamination and degradation, etc. problems, to achieve the effect of satisfying cloning and high-throughput sequencing, high specificity, and avoiding cross-contamination

Active Publication Date: 2020-11-17
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 will affect the accuracy of eDNA analysis results and the high cost of experiments, and propose a fish diversity method based on eDNA Quantitative method and its sampling and filtering device

Method used

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  • A Quantitative Method of Fish Diversity Based on Edna and Its Sampling and Filtering Device
  • A Quantitative Method of Fish Diversity Based on Edna and Its Sampling and Filtering Device
  • A Quantitative Method of Fish Diversity Based on Edna and Its Sampling and Filtering Device

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: a kind of quantitative method and its sampling filtration device based on eDNA fish diversity, comprise the following steps:

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

[0039] S2. Use a sterilized eDNA-based quantitative sampling and filtration device for fish diversity to filter the collected samples, remove the microporous filter membrane after filtration, 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 and grind it, and pour the debris into a 2mL centrifuge tube for future use;

[0041] S4, take out the debris of the sample filter membrane, and use D...

Embodiment 2

[0053] Example 2: Based on Example 1, the difference is that the 7 sampling points in the upper and lower reaches of the Huairou Reservoir were intensively sampled, the sampling volume was 1L, and the collected samples used a sterilized eDNA-based fish Quantitative sampling and filtration device with various types of diversity, the filter membrane is placed in a liquid nitrogen mortar and crushed, and the DNA extraction experiment is carried out within 24 hours. The extracted DNA samples are put into an ultra-micro spectrophotometer for concentration detection, and the most covered species are selected Identify the universal primers with the highest accuracy, use the primers for diverse PCR amplification, take 2uL of the purified PCR product and connect it to the carrier, transform competent cells, and culture at a constant temperature at 37°C overnight. After cloning, pick 50 single colonies and use The primers were verified by colony PCR. The PCR product was detected by agaro...

Embodiment 3

[0055] Embodiment 3: Based on Examples 1 and 2, the difference is that 4 sampling points with the gene sequence of the broadfin carp are selected for sampling, and the collected samples are subjected to a DNA extraction experiment. According to the gene sequence of the BLAST comparison result, in Download the full gene sequence of Zaccoplatypus from the NCBI database, perform primer screening in the Primer5.5 software, obtain the upstream and downstream primer fragments of the target gene, and perform primer synthesis, using the collected samples as templates, using the target gene Primers are used for PCR amplification, and the recovered PCR products are subjected to TA cloning, and the target colonies are picked for plasmid extraction. The extracted plasmid is used as an absolute quantitative standard. The RealTimePCR reaction system is configured according to the DNA sample, and the RealTimePCR amplification experiment is carried out. Each sample is 3 repeated tests. Synthe...

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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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Patent Type & Authority Patents(China)
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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