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
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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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