Method for enriching and detecting lake and reservoir planktonic viruses based on environmental DNA technology and application of method

Abstract

The invention discloses a method for enriching and detecting lake and reservoir planktonic viruses based on an environmental DNA technology and application of the method, and belongs to the technical field of microorganism detection.The method comprises the steps that 1, according to the change rate of dissolved oxygen on the vertical section of a lake and reservoir water body, the water body is divided into a lake upper layer, an oxygen spring layer and a lake lower layer; 2, when the water depth is smaller than 3 m and thermal stratification and oxygen stratification do not exist, collecting a water sample at the position 0.5 m away from the surface layer, when the water depth is 3-10 m, collecting the water sample at the position 0.5 m away from the surface layer and the water sample at the position 1-2 m away from the upper portion of bottom sediment, and when the water depth is larger than 10 m, at least collecting samples of three water layers; according to the method, by combining two different sample treatment methods, lake and reservoir planktonic virus communities including intracellular viruses and virions can be more comprehensively detected, so that more complete information is obtained. By knowing the structure and function of the lake and reservoir planktonic virus community, a scientific basis is provided for water environment monitoring and management, water body pollution can be prevented and controlled, and the water ecological environment is protected.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and specifically to a method for enriching and detecting lake and reservoir plankton viruses based on environmental DNA technology and its application. Background Art

[0002] Plankton viruses are the general term for various groups of viruses suspended in water bodies, with phytoplankton, zooplankton, planktonic bacteria, etc. as hosts. Plankton viruses can regulate the species diversity, population distribution, and community structure of microorganisms by lysing the dominant populations in the water body microbial community, thereby affecting the cycling of elements such as carbon and nitrogen.

[0003] Environmental DNA technology is to extract DNA from environmental media (water, soil, sediment, etc.), perform PCR amplification and sequencing on specific DNA fragments of the genome, so as to achieve qualitative and quantitative analysis of biological communities. However, due to the preference of water sample pretreatment methods, the samples collected by different methods are different, which will lead to incomplete detection results of the virus community and cannot truly and comprehensively reflect the information of the water environment plankton virus community.

[0004] In early studies, tangential flow filtration was used to concentrate a large number of plankton viruses from natural water bodies, but tangential flow filtration has problems such as expensive equipment, long time consumption, poor stability, and high requirements for the technical level of operators. Chemical flocculation technology is also widely used in water sample pretreatment. It was first used in wastewater treatment technology and can effectively remove virus particles in wastewater. Later, it was applied to the enrichment of marine virus samples. The enriched samples can significantly increase the proportion of virus information annotation, but will lose the viral genomes in cells.

[0005] There are usually thermocline and oxycline phenomena in deep lakes and reservoirs, including the epilimnion, thermocline (oxycline), and hypolimnion. There are significant differences in the plankton communities in different water layers. However, previous studies on water plankton viruses mainly focused on the surface water body and less on stratified water bodies. For this reason, a method for enriching and detecting lake and reservoir plankton viruses based on environmental DNA technology and its application are proposed.

[0006] The above information disclosed in this background art is only used to increase the understanding of the background art of the present invention. Therefore, it may include prior art that is not known to those of ordinary skill in the art. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. For this reason, an object of the present invention is to propose a method for enriching and detecting lake and reservoir plankton viruses based on environmental DNA technology and its application.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] Method and application for enriching and detecting lake and reservoir planktonic viruses based on environmental DNA technology, comprising the following steps:

[0010] Step 1: Divide the water body into the epilimnion, thermocline and hypolimnion according to the change rate of dissolved oxygen in the vertical section of the water body;

[0011] Step 2: When the water depth is less than 3 m and there is no thermal stratification and oxygen stratification, collect water samples at 0.5 m below the surface. When the water depth is 3 - 10 m, collect water samples at 0.5 m below the surface and at 1 - 2 m above the bottom sediment respectively. When the water depth is greater than 10 m, collect samples from at least 3 water layers, including water samples at 0.5 m in the epilimnion, water layers in the thermocline, and water samples at 1 - 2 m above the sediment in the hypolimnion;

[0012] Step 3: Use a 200 μm nylon sieve to pre-filter to remove large particulate matter and large plankton in the water body, and use a polycarbonate filter membrane with a pore size of 0.22 μm to filter microorganisms. After filtration, remove the filter membrane and wrap it with aluminum foil, then put it into a sterilized 2 mL centrifuge tube for storage. Collect 3 - 6 duplicate samples by filtration for each sample. The samples obtained in the range of 0.22 - 200 μm are intracellular virus samples, and collect the filtered water samples generated in Step 3 for collecting virion samples;

[0013] Step 4: Add an appropriate amount of 10 g / L FeCl3 solution to the filtered water according to the ratio of adding 100 μL FeCl3 solution per liter of filtered water, mix well and let it stand in the dark for 1 h. Use a polycarbonate filter membrane with a pore size of 0.8 μm to filter at least 5 L of water body. After filtration, remove the filter membrane and wrap it with aluminum foil, then put it into a sterilized 2 mL centrifuge tube for storage. The samples obtained by enrichment with FeCl3 are virion samples, and finally obtain intracellular virus and virion samples;

[0014] Step 5: Obtain planktonic virus community samples along the lake and reservoir water column using two treatment methods (filtration with pore sizes > 0.22 μm and < 0.22 μm). The former focuses on intracellular viruses and the latter focuses on virions. Therefore, combining the two methods can more comprehensively detect lake and reservoir planktonic viruses.

[0015] As a further optimization scheme of the present invention, in Step 2, at least 10 L of water samples are collected at each sampling point.

[0016] As a further optimization scheme of the present invention, in Step 3, the volume of the filtered water sample for eutrophic water bodies is 100 - 500 mL, the volume of the filtered water sample for mesotrophic water bodies is 500 - 1000 mL, and the volume of the filtered water sample for oligotrophic water bodies is greater than 1000 mL.

[0017] As a further optimized solution of the present invention, in step three, the filtration time of each independent filter membrane sample needs to be greater than 30 minutes.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] In the present invention, by combining two different sample processing methods, the planktonic virus communities in lakes and reservoirs can be more comprehensively detected, including intracellular viruses and virions, so as to obtain more complete information. And it is applied to different types of lakes and reservoirs, including deep lakes, shallow lakes, reservoirs, etc. The method used is simple and easy to operate, and the cost of the required equipment and materials is relatively low, which can reduce the detection cost. By understanding the structure and function of the planktonic virus communities in lakes and reservoirs, it provides a scientific basis for water environment monitoring and management, helps prevent and control water pollution, and protects the water ecological environment.

[0020] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will become apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a flowchart of the enrichment sampling and processing of planktonic viruses in water bodies based on the environmental DNA technology of the present invention;

[0022] Figure 2 is a graph showing the analysis results of the common vOTUs of planktonic viruses in a reservoir in an application example of the present invention;

[0023] Figure 3 is a graph showing the results of the NMDS analysis and ANOSIM inter-group difference significance test of the planktonic virus community in a reservoir in an application example of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] Embodiment 1

[0026] As shown in the attached Figure 1 figure, a method for enriching and detecting planktonic viruses in lakes and reservoirs based on the environmental DNA technology includes the following steps:

[0027] Determine sampling sites: Determine the number of sampling points according to the water area, shape, ecological distribution characteristics of microorganisms, and investigation purposes of lakes and reservoirs, and select representative locations that can reflect the basic situation of the microorganisms in the entire water body. For lakes, sampling points should be set near the shore and in the middle. Sampling points can be scattered and selected in the center of the lake, the center of large lake bays, near the inlet and outlet, and shallow coastal waters (with and without aquatic plants) according to the shape of the lake; for reservoirs, sampling points should be set in the center of the reservoir (for river-type reservoirs, in the center of the upper, middle, and lower reaches), the center of large reservoir bays, the main inlet, outlet, and the confluence of the incoming rivers.

[0028] Use a depth sounder to measure the water depth, and use a multi-parameter water quality analyzer to measure the vertical profiles of water temperature and dissolved oxygen at the sampling stations. According to the change rate of dissolved oxygen in the vertical profile, the water body is divided into the epilimnion, the oxycline (the water layer with a sharp change in dissolved oxygen), and the hypolimnion. If the water body is shallow (water depth less than 3 m) and there is no oxygen stratification, collect water samples from the surface layer (0.5 m). When the water depth is 3–10 m, collect water samples from the surface layer (0.5 m) and the bottom layer (1–2 m upward from the sediment surface). When the water depth is greater than 10 m, generally collect water samples from at least 3 water layers, including water samples from the epilimnion (0.5 m), the oxycline, and the hypolimnion (1–2 m upward from the sediment surface).

[0029] Collection of water body samples: After clarifying the sampling stations and sampling water layers, use a 5L water sampler or a water pump to collect water samples from different water layers. At least 10L of water samples should be collected at each sampling point and filled into polyethylene plastic buckets pre-written with sample numbers. Before filling the water samples, rinse them 3 times with in-situ water samples at the corresponding depth to prevent sample contamination. The collected water samples should be transported back to the laboratory within 1h for immediate processing, or stored in a vehicle-mounted low-temperature refrigerator at 4°C and transported back to the laboratory as soon as possible.

[0030] Collection of intracellular virus samples: Use ultrapure water to clean the filtration equipment including filter cups, filter elements, measuring cups, graduated cylinders, and suction flasks, and assemble the filtration device. Mix the raw water in the polyethylene plastic bucket up and down evenly, and pre-filter it with a nylon sieve with a pore size of 200μm to remove large particulate matter and large plankton in the water body. Then, filter it with a polycarbonate filter membrane with a pore size of 0.02μm (diameter 47mm, Millipore).

[0031] Start the vacuum pump, adjust the air pressure in the suction filtration system to 0.02 MPa. After rinsing the measuring cup and graduated cylinder with the original water, add the water sample in small amounts and multiple times. First, filter a small amount of the original water to rinse the suction flask and then pour it out. Each time a sample from a different water layer is changed, the suction flask needs to be rinsed again to prevent sample contamination and avoid external contamination. Each time the sample is added, it should be no less than 50 mL. For each independent filter membrane sample, ensure that the filtration time is greater than 30 min to ensure sufficient biomass is collected. For water bodies with relatively severe eutrophication, the volume of the filtered water sample is about 100–500 mL; for mesotrophic water bodies, the volume of the filtered water sample is approximately 500–1000 mL; for oligotrophic water bodies, the volume of the filtered water sample can exceed 1000 mL.

[0032] After filtration is completed, cut the aluminum foil to an appropriate size. Use forceps to hold the white edge of the filter membrane and transfer the filter membrane to the center of the aluminum foil. Fold the aluminum foil into a long strip and place it in a sterilized 2 mL centrifuge tube for storage. Write information such as the sample number, filtration time, filtration volume, filter membrane pore size, and the number of filter membranes in the tube on the outer wall of the centrifuge tube. During this process, the tip of the forceps is strictly prohibited from touching the sample area in the center of the filter membrane. When folding the aluminum foil, ensure that the sample area in the center of the filter membrane is not contaminated by the aluminum foil. If sample contamination or filter membrane breakage occurs, the sample is invalidated. Collect 3–6 replicate samples for each sample. Before filtering the water samples of different samples each time, the filter cup, filter element, measuring cup, and graduated cylinder must be rinsed with ultrapure water 3 times to avoid cross-contamination between samples. After filtration, use ultrapure water to clean the glass sand core filtration device, suction flask, etc. and let them air dry naturally to prevent bacterial growth and contamination.

[0033] Collection of virion samples: Weigh 2.415 g of ferric chloride hexahydrate (FeCl3·6H2O) and place it in a 50 ml sterile centrifuge tube. Add 50 ml of sterilized water to fully dissolve the ferric chloride hexahydrate, and finally prepare a 10 g / L FeCl3 solution, which is stored at 4 °C. When the solution becomes turbid and precipitates, discard the solution, and do not dilute the solution to prevent the formation of ferric hydroxide precipitate.

[0034] Pour the water filtered through a 0.22 μm pore size filter membrane into a sterilized plastic bucket. Collecting 5 L of filtered water can meet the needs of subsequent experiments. Add 500 μL of FeCl3 solution, mix well, and let it stand in the dark for 1 h.

[0035] Clean the filtration equipment, including the filter cup, filter element, measuring cup, graduated cylinder, and suction flask, with ultrapure water, and assemble the filtration device. Place a 0.8 μm pore size polycarbonate filter membrane (diameter 47 mm, Millipore) in the center of the glass sand core filter element, fix and clamp it with the supporting filter cup, and then start the vacuum pump. After the air pressure in the suction filtration system drops to 0.02 MPa, add the pre-treated filtered water. Ensure that the filtration time for each independent filter membrane sample is greater than 30 min. During the filtration process, if the filter membrane becomes blocked and the filtration efficiency is too low, the filter membrane can be replaced.

[0036] After filtration, cut the aluminum foil to an appropriate size. Use forceps to hold the white edge of the 0.8-μm pore size filter membrane and transfer the filter membrane to the center of the aluminum foil. Fold the aluminum foil into a long strip and place it in a sterilized 2-ml centrifuge tube for storage. During this process, it is strictly prohibited for the tip of the forceps to touch the sample area in the center of the filter membrane. When folding the aluminum foil, ensure that the sample area in the center of the filter membrane is not contaminated by the aluminum foil. If sample contamination or filter membrane breakage occurs, the sample is invalidated. The centrifuge tube should be clearly marked with information such as the sample number, filtration time, filtration volume, filter membrane pore size, and the number of filter membranes in the tube. The writing should be clear. Store all filter membranes in an ultra-low temperature freezer for subsequent DNA extraction and sequencing.

[0037] Application Example

[0038] Taking Shidou Reservoir in Xiamen City, Fujian Province and Shanmei Reservoir in Quanzhou City as examples, sampling points were set at the deepest parts in front of the dams of the two reservoirs. A total of 24 samples were collected, including 2 sampling stations, 3 water layers, 2 different seasons, and 2 sample processing methods.

[0039] As shown in the appendix Figure 2 As shown, about 70% of the viral taxonomic units vOTUs of the two sample processing methods are unique, indicating that there are significant differences in the vOTUs collected by the two methods.

[0040] As shown in the appendix Figure 3 As shown, the results of NMDS analysis and ANOSIM test indicate that there are significant differences in the composition of the planktonic virus community, and there are significant differences in the planktonic viruses between the two sample processing methods and the two reservoirs.

[0041] The above results show that there is a 70% difference in the number of planktonic virus species collected by the two sample processing methods, and the community compositions are significantly different. Combining the two methods can more comprehensively detect the planktonic virus community in lakes and reservoirs and is applicable to different natural water bodies.

[0042] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. In addition, without conflict, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0043] In the attached drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments are involved. Other structures can refer to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0044] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for enriching and detecting planktonic viruses in lakes and reservoirs based on environmental DNA technology and its application, characterized in that: The following steps are involved: Step 1: According to the change rate of dissolved oxygen in the vertical section of the water body, the water body is divided into the upper lake layer, the oxygen jump layer and the lower lake layer; Step 2: When the water depth is less than 3m and there is no thermal stratification and oxygen stratification, collect water samples at 0.5m from the surface. When the water depth is 3-10m, collect water samples at 0.5m from the surface and 1-2m above the bottom sediment. When the water depth is greater than 10m, collect samples from at least three water layers, including water samples at 0.5m above the lake, the oxygen cline, and 1-2m above the sediment in the lower lake. Step 3: Use 200 μm nylon mesh to pre-filter to remove large particles and large plankton in the water, and use a polycarbonate filter membrane with a pore size of 0.22 μm to filter microorganisms. After filtration, remove the filter membrane and wrap it with aluminum foil, and store it in a sterilized 2 mL centrifuge tube. Filter and collect 3-6 duplicate samples for each sample. The samples obtained in the range of 0.22-200 μm are intracellular virus samples. Collect the filtered water sample produced in step 3 for collecting virus body samples; Step 4: Add an appropriate amount of 10g / LFeCl3 solution to the filtered water at a ratio of 100μL FeCl3 solution per liter of filtered water, mix thoroughly and place in the dark for 1h, filter at least 5L of water using a polycarbonate filter membrane with a pore size of 0.8μm, remove the filter membrane after filtration and wrap it with aluminum foil, and store it in a sterilized 2mL centrifuge tube. The sample obtained by FeCl3 enrichment is a virion sample, and finally intracellular virus and virion samples are obtained; Step 5: Samples of the floating viroplankton community were obtained along the lake water column using two treatment methods (>0.22μm and <0.22μm pore size filtration), the former focusing on intracellular viruses and the latter focusing on virions.

2. The method for enriching and detecting planktonic viruses in lakes and reservoirs based on environmental DNA technology according to claim 1 and its application, characterized in that: In step 2, at least 10L of water sample is collected at each sampling point.

3. The method for enriching and detecting planktonic viruses in lakes and reservoirs based on environmental DNA technology according to claim 1 and its application, characterized in that: In step three, the volume of filtered water samples for water bodies with severe eutrophication is 100–500 mL, the volume of filtered water samples for water bodies with mesotrophication is 500–1000 mL, and the volume of filtered water samples for water bodies with oligotrophication is greater than 1000 mL.

4. The method for enriching and detecting planktonic viruses in lakes and reservoirs based on environmental DNA technology according to claim 1 and its application, characterized in that: In step 3, the filtration time of each independent membrane sample must be greater than 30 minutes.

Images