JP7507082B2 - Environmental DNA sample collection device and environmental DNA sample collection method - Google Patents

Description

The present invention relates to an environmental DNA sample collection device and an environmental DNA sample collection method.

When assessing the environmental impact of construction work or selecting suitable sites for mitigation, the distribution of biological species and their changes are important information. One method of obtaining biological information in water areas is environmental DNA analysis, which exists in the soil, water, and other environments. Environmental DNA analysis obtains information on biological species by detecting DNA from minute biological fragments and excrement floating in the water (environment) of the surveyed water area. By analyzing environmental DNA, it is possible to identify the biological species that inhabit the area and estimate their quantity.
A typical environmental DNA analysis involves collecting about 1 liter of water (environmental water) on-site as a sample, filtering the sample with, for example, a filter paper with a pore size of 0.7 μm, and subjecting the DNA extracted from the residue captured on the filter paper to real-time PCR analysis or amplicon analysis using a next-generation sequencer. Because the analysis requires filtering a large amount of water and filtering takes time, the large amount of water collected on-site is sometimes stored in a cooler box or the like and brought back to an analysis facility for filtering and analysis.
Conventionally, as a sampling method and device for sampling a liquid to be measured on-site, the technology disclosed in Patent Document 1 is known. The sampling method and device of Patent Document 1 includes a filter for removing unnecessary matter that separates the inside of a sampling container into upper and lower parts, and samples liquid introduced to the upper side of the sampling container through the filter.

JP 2003-344229 A

The sampling method and device of Patent Document 1 simply collects liquid introduced into the upper part of a sampling container through a filter on-site, but does not capture the sample contained in the liquid with a filter. Therefore, this sampling method and device alone cannot analyze environmental DNA, and does not reduce labor.
In addition, the concentration of environmental DNA dispersed is low, so a large amount of water needs to be collected from the sampling point in the surveyed water area in order to sample for analysis. In particular, in surveyed water areas with rivers or ocean currents, if water is collected continuously according to the filtration capacity, sample water that has passed the measurement timing will be mixed in, causing a deterioration in the quality of the collected sample.
The present invention aims to solve the above-mentioned problems, and to provide an environmental DNA sample collection device and an environmental DNA sample collection method that are excellent in obtaining biological information in surveyed water areas and reduce the labor required for analysis.

In order to solve the above problems, the present invention provides an environmental DNA sample collection device to be installed in an environmental DNA survey water area. The environmental DNA sample collection device includes a storage container for sample water , a filtration device , a wastewater storage container, and a filtration pump , and collects a required amount of sample water from a survey water area during collection and stores it in the storage container , supplies the sample water stored in the storage container to the filtration device by the filtration pump , and filters it with the filtration device to collect an environmental DNA sample . The wastewater storage container is configured to temporarily store filtrate after filtering with the filtration device. The filtration pump sucks gas from the inner space of the wastewater storage container to create a negative pressure state within the wastewater storage container, thereby accelerating the drainage of filtrate, and the sample water stored in the storage container is continuously supplied to the filtration device.
In the present invention, the required amount of sample water can be collected at a time from a specified water collection point in the surveyed water area and stored in a storage container. Therefore, the required amount of sample water can be quickly sampled even in a surveyed water area with a river or ocean current. Then, the collected sample water is discharged into the water area through a filtration device, so that fine biological fragments and excrement contained in the water (environment) of the surveyed water area can be captured by the filtration device. In other words, the environmental DNA sample required for analysis can be suitably collected while adjusting for the long filtration time. Therefore, it is possible to obtain biological information in the surveyed water area and perform appropriate environmental DNA evaluation. Moreover, instead of a storage container containing the sample water, the sample captured by the filtration device can be brought back to an analysis facility, etc., so that the labor required for analysis can be suitably reduced.

It is also preferable that the storage container and the filtration device are combined to form one filtration set, and that the filtration sets are provided in the number required for the number of collections.
By configuring in this way, the required amount of sample water can be collected in the storage container of each filtration set at each sampling time, while the environmental DNA samples required for analysis can be collected individually by each filtration device. Therefore, the labor involved in analyzing environmental DNA can be further reduced. In addition, since the filtration sets are provided in the number required for the number of collections, the influence of the sample water collected previously can be eliminated. In addition, in the case of water quality that makes it difficult to filter a large amount of sample water, the amount of sample water that has been filtered can be understood by checking the amount of sample water remaining in the storage container (remaining amount). Furthermore, by replacing the filtration set with a new one when collecting it, the environmental DNA samples required for analysis can be continuously collected.

In addition, it is preferable that at least the filtration device is placed in a temperature-controlled environment.
By installing a filter in a controlled environment (e.g., an environment controlled at a refrigeration or freezing temperature), deterioration of minute biological fragments and excrement captured by the filter can be prevented, allowing accurate biological information to be obtained in the water area, which allows for appropriate evaluation of environmental DNA.

The environmental DNA collection method of the present invention involves installing an environmental DNA sample collection device in an environmental DNA survey water area to sample environmental DNA. The environmental DNA sample collection method includes a collection device installation step of installing an environmental DNA sample collection device in a survey water area, the environmental DNA sample collection device including one filtration set formed by combining the storage container and the filtration device, the number of which corresponds to a plurality of preset sampling times, a water collection step of collecting and storing sample water in the storage container of the corresponding filtration set in accordance with the sampling time, a filtration step of supplying sample water from the storage container to the filtration device and filtering it, and discharging the filtrate after filtration, and a recovery step of recovering the filtration residue as a sample for environmental DNA analysis.
In the present invention, the sampling device installation step installs an environmental DNA sample sampling device in the surveyed water area, and the water sampling step allows sample water to be sampled at each sampling time and stored in the storage container of each filtration set. In other words, the required amount of sample water collected at a certain specified water sampling location and sampling time can be secured in the storage container. Therefore, the required amount of sample water can be sampled quickly even in a real sea area with a flow. Then, the filtration step allows the sample water in the storage container to be discharged into the water area through the filtration device, so that fine biological fragments and excrement contained in the water (environment) of the surveyed water area can be captured by the filtration device. Then, the recovery step allows the filtration residue to be recovered as a sample for environmental DNA analysis, so that biological information in the surveyed water area can be obtained easily and appropriate environmental DNA can be evaluated. Moreover, the filtration residue can be recovered on-site in the surveyed water area, so that the labor required for analysis can be suitably reduced.

The environmental DNA sample collection device and method of the present invention are excellent for obtaining biological information in surveyed water areas and can reduce the labor required for analysis.

1 is a schematic diagram showing the installation mode of an environmental DNA sample collection device according to an embodiment of the present invention in a survey water area. FIG. 2 is a block diagram showing the components of an environmental DNA sample collection device according to an embodiment of the present invention. A configuration diagram showing other components of the environmental DNA sample collection device according to an embodiment of the present invention. 10 is a schematic diagram showing another installation mode of an environmental DNA sample collection device according to an embodiment of the present invention in a surveyed water area. FIG. 10 is a schematic diagram showing another installation mode of an environmental DNA sample collection device according to an embodiment of the present invention in a surveyed water area. FIG.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings as appropriate. Fig. 1 is a schematic diagram showing an installation state of an environmental DNA sample collection device according to an embodiment of the present invention in a survey water area, and Fig. 2 is a configuration diagram showing components of the environmental DNA sample collection device according to an embodiment of the present invention.
The environmental DNA sample collection device M of this embodiment is installed on the structural body 110 of a floating structure 100 installed in the survey water area W (water collection location) as shown in Figure 1, and is a device that collects samples for environmental DNA analysis directly in the survey water area (site) W.
The structure body 110 is a raft-like structure having buoyancy, and is moored via a wire 101 and an anchor 102 so as to remain on the sea surface of the surveyed water area W. The floating structure 100 can be moved on the sea surface by a drive mechanism (not shown) provided on the floating structure 100 or by being towed by a ship, or the like.

As shown in Figure 2, the environmental DNA sample collection device M comprises a water sampling pump 10, a group of storage containers 20, a group of filtration devices 30, a wastewater storage container 40, a filtration pump 50, a control unit 60, a battery 70, and a solar panel (power generation equipment) 80.
The water sampling pump 10 is a pump for sampling seawater from the surveyed water area W and sending it to the downstream storage container group 20. The water sampling pump 10 is operated under the control of the control unit 60 using power from a battery 70.
A water sampling pipe 11 is connected to the suction side of the water sampling pump 10. As shown in Fig. 1, the water sampling pipe 11 is supported by a support member 120 extending from a structure body 110 toward the sea and is submerged in the sea. The water sampling port 11a of the water sampling pipe 11 is installed, for example, at a predetermined depth from the sea surface. Note that a water sampling port moving mechanism (not shown) or the like may be provided at the water sampling port 11a so that the water sampling port 11a faces upstream in the seawater flow direction.
As shown in Fig. 2, a discharge pipe 12 is connected to the discharge side of the water sampling pump 10. The discharge pipe 12 is connected to a branch pipe 13 at the rear stage. A first on-off valve 12a for opening and closing the discharge pipe 12 is provided in the middle of the discharge pipe 12. The first on-off valve 12a is operated under the control of the control unit 60 using power from a battery 70. A plurality of inlet pipes 14 leading to the storage container group 20 are connected to one end of the branch pipe 13. A second on-off valve 14a for opening and closing each inlet pipe 14 is provided in the middle of each inlet pipe 14. The second on-off valve 14a is operated under the control of the control unit 60 using power from the battery 70.
The other end of the branch pipe 13 extends to the wastewater storage container 40 and opens into the wastewater storage container 40. A third opening/closing valve 13a for opening and closing the branch pipe 13 is provided in the middle of the branch pipe 13 leading to the wastewater storage container 40. The third opening/closing valve 13a is operated under the control of the control unit 60 using power from the battery 70.

In this embodiment, the storage container group 20 is installed in a building 20a separated by a partition wall. The storage container group 20 is not limited to being installed in the building 20a, and may be configured to be covered with a light-shielding material. The storage container 21 itself described later may be formed of a material having light-shielding properties to configure the storage container group 20. Each of the multiple storage containers 21 constituting the storage container group 20 has a volume capable of storing the amount of sample water required for analyzing environmental DNA, for example, a volume of 20 liters or more. For example, a replaceable water bag is used as the storage container 21. The storage containers 21 are installed in the number that can individually store the sample water collected at each preset sampling time. For example, in the case of sampling 12 times in total every two hours, at least 12 storage containers 21 are provided in the building 20a. An inlet pipe 14 branched from a branch pipe 13 is connected to the top of each storage container 21. An outlet pipe 15 is connected to the bottom of each storage container 21. The downstream end of each outlet pipe 15 leads to the inside of the refrigerator compartment 35 of the filtration device group 30. A fourth on-off valve 15a for opening and closing the outlet pipe 15 is provided midway along each outlet pipe 15. The fourth on-off valve 15a is operated under the control of the control unit 60 using power from the battery 70. The flow rate of the sample water flowing through the outlet pipe 15 is set by the amount of water sucked in by the filtration pump 50, which will be described later.

The filtering device group 30 includes a plurality of filtering devices, each including a first filter 31 disposed midway downstream of each outlet pipe 15, and a second filter 32 disposed after the first filter 31. Each first filter 31 serves to remove larger particles and unwanted matter contained in the sample water that has flowed through the outlet pipe 15. In this embodiment, the first filter 31 has a mesh size of 100 μm.
Each second filter 32 filters the sample water after the removal of dust and unnecessary substances, and serves to capture fine biological debris, excrement, and the like contained in the sample water. In this embodiment, a filter with an opening size of 7 μm is used as the second filter 32. Each second filter 32 is disposed in the refrigerator chamber 35, and can be removed from the refrigerator chamber 35 after filtration.
The refrigerator compartment 35 is cooled by a cooling device 33 that is powered by a battery 70. The inside of the refrigerator compartment 35 is shielded from light, providing an environment in which deterioration of biological debris, excrement, and the like captured by filtration is unlikely to progress.
The filtered water after being filtered by each second filter 32 is discharged through the individual drain pipe 16b of the subsequent drain pipe 16. The downstream end of the drain pipe 16 is connected to the other end of the branch pipe 13. A fifth opening/closing valve 16a for opening and closing the individual drain pipe 16b is provided midway along each individual drain pipe 16b. The fifth opening/closing valve 16a is operated under the control of the control unit 60 using power from the battery 70.
In this embodiment, a single filtration set is configured by combining the storage container 21 and the filtration device. At least the number of filtration sets required for the number of collections (samplings) is provided. For example, in the case of sampling 12 times in total every two hours, at least 12 filtration sets are provided.

The wastewater storage container 40 is a container that temporarily stores wastewater flowing in through the branch pipe 13. The discharge pipe 17 is connected to the bottom of the wastewater storage container 40. As shown in FIG. 1, the discharge pipe 17 is supported by a support member 130 that extends from the structure body 110 toward the sea, and is submerged in the sea at a position away from the water sampling port 11a on the opposite side. In this embodiment, the discharge port 17b of the discharge pipe 17 is located at a position deeper than the water sampling port 11a so as not to affect the sample water sampled from the water sampling port 11a. A sixth opening/closing valve 17a that opens and closes the discharge pipe 17 is provided in the middle of the discharge pipe 17. The sixth opening/closing valve 17a is operated under the control of the control unit 60 using power from the battery 70.
A suction pipe 18 connected to a filtration pump 50 is attached to the top of the wastewater storage container 40. A suction port 18a of the suction pipe 18 opens into the inner space A above the water level H1 of the wastewater stored in the wastewater storage container 40.
The filtration pump 50 sucks gas from the inner space A of the wastewater storage container 40 via the suction tube 18, creating a negative pressure state inside the wastewater storage container 40, thereby accelerating drainage in the branch pipe 13 and promoting the filtration action. The filtration pump 50 is operated under the control of the control unit 60 using power from the battery 70. The suction amount of the filtration pump 50 is set so that the sample water stored in the storage container 21 flows into the second filter 32 over a period of time.

The control unit 60 controls the operation of the water sampling pump 10, the first on-off valve 12a, the second on-off valve 14a, the third on-off valve 13a, the fourth on-off valve 15a, the fifth on-off valve 16a, and the sixth on-off valve 17a, and also controls the operation of the filtration pump 50. The control unit 60 is an ECU (Electronic Control Unit) equipped with a CPU, RAM, ROM, and input/output circuits. Various controls by the control unit 60 are realized by reading a program pre-stored in the ROM into the RAM and executing it with the CPU. The above-mentioned operation controls can also be performed manually.

The battery 70 is charged with power supplied from the solar panel 80 and supplies power to each of the above-mentioned components. The solar panels 80 are panels for generating electricity using sunlight, and a predetermined number of solar panels are installed on the structure body 110 so that the battery 70 is charged with the power required to capture environmental DNA. It is preferable that the battery 70 is configured to be replaceable. By configuring it to be replaceable, for example, if the battery 70 is not sufficiently charged due to weather or the like, it can be replaced with a pre-charged battery 70 when collecting samples, etc.

Next, a detailed description will be given of a method for collecting environmental DNA samples using the environmental DNA sample collection device M. Below, a description will be given of a method for collecting environmental DNA samples in which a floating structure 100 is placed in a surveyed water area W of the ocean, but the surveyed water area W is not limited to the ocean, and also includes lakes, marshes, and rivers.
The method for collecting environmental DNA samples includes a collection device installation step, a water collection step, a filtration step, and a recovery step. Before the water is collected, the water collection pump 10 and the filtration pump 50 are stopped, and each opening/closing valve is closed.
The sampling device installation process is a process of installing an environmental DNA sample sampling device M in the investigation water area W. In the sampling device installation process, the floating structure 100 is moved to the investigation water area W by autonomous navigation using a drive mechanism (not shown) or by towing by a ship. Once the floating structure 100 has been moved to the investigation water area W, multiple anchors 102 are lowered through a wire 101 to moor the floating structure 100 in the investigation water area W.

The water sampling step is a step of collecting and storing sample water in the storage container 21 of the corresponding filtration set at a preset sampling time.
Specifically, in the water sampling process, a few minutes before the filtration start time, the water sampling pump 10 is operated to pass the sampled water through the water sampling pipe 11, the discharge pipe 12, etc., thereby performing co-washing. In this case, the control unit 60 operates the water sampling pump 10 and opens the first on-off valve 12a of the discharge pipe 12 and the third on-off valve 13a of the branch pipe 13. This causes the water sampling pipe 11, the discharge pipe 12, and the branch pipe 13 to be co-washed. The water after the co-washing is drained from the branch pipe 13 to the drainage storage container 40, and is discharged from the discharge pipe 17 by opening the sixth on-off valve 17a by the control unit 60.
Thereafter, when the filtration start time arrives, the control unit 60 opens the second on-off valve 14a of the inlet pipe 14 leading to the storage container 21 of the corresponding filtration set, and closes the third on-off valve 13a of the branch pipe 13. This causes a predetermined amount of sample water to be stored in the storage container 21 of the corresponding filtration set. The control unit 60 then stops the water sampling pump 10. The stop control of the water sampling pump 10 can be performed, for example, by detecting that a predetermined amount of water has been collected in the storage container 21 using a liquid level sensor or the like.

The filtration step is a step of supplying sample water from the storage container 21 to the filtration device, filtering the sample water, and discharging the filtered water. In the filtration step, the control unit 60 opens the fourth on-off valve 15a of the outlet pipe 15 leading to the storage container 21 of the corresponding filtration set, opens the fifth on-off valve 16a of the individual drain pipe 16b leading to the corresponding filtration set, and operates the filtration pump 50. The sixth on-off valve 17 of the discharge pipe 17 is kept in a closed state. Then, the sample water flows from the storage container 21 through the outlet pipe 15 to the first filter 31 of the filtration device, and larger debris and other unwanted substances contained in the sample water are removed by the first filter 31. The debris and other unwanted substances removed by the first filter 31 are discarded.
Thereafter, the sample water filtered by the first filter 31 is introduced into the refrigeration chamber 35 and filtered by the second filter 32. As a result, the second filter 32 captures minute biological debris, excrement, and the like contained in the sample water.
The control unit 60 controls the filtration to stop when a preset time has elapsed after the start of filtration, for example, 1 hour or 30 minutes. In other words, the control unit 60 closes the fourth on-off valve 15a of the outlet pipe 15 and the fifth on-off valve 16a of the individual drain pipe 16b after the predetermined time has elapsed, and stops the filtration pump 50.
The filtered water after being filtered by the second filter 32 is discharged through the individual drain pipe 16b of the subsequent drain pipe 16, flows into the branch pipe 13, and is then discharged into the wastewater storage container 40. Then, the control unit 60 opens the sixth opening/closing valve 17a of the discharge pipe 17 to discharge the filtered water discharged into the wastewater storage container 40. This causes the filtered water to be discharged into the sea from the discharge port 17b through the discharge pipe 17.
If the sample water contains a large amount of suspended solids and it is not possible to filter all of the sample water in the storage container 21, the filtration may be stopped midway.

The recovery step is a step of recovering the filtration residue captured by the second filter 32 as a sample for environmental DNA analysis. The second filter 32 may be removed from the refrigerator chamber 35 for each filtration set, or a plurality of filtration sets may be removed from the refrigerator chamber 35 together. When the second filter 32 is removed immediately after filtration is stopped, it is preferable to provide a waiting time (e.g., 30 seconds) at least after filtration is stopped so that the sample water remaining in the second filter 32 is filtered.
The recovered second filter 32 is preferably stored in a frozen environment, for example, at −20° C. Furthermore, when the second filter 32 is recovered within 24 hours after the filtration is stopped, it is preferable to keep the refrigerator chamber 35 in a cool, dark environment, for example, at 4° C.
Furthermore, when recovering the second filter 32, it is preferable to check the amount of sample water remaining in the storage container 21 and grasp the amount of filtration.
In the water sampling method as described above, it is preferable that the control unit 60 starts the water sampling process in the other filtration set, for example, two hours after the filtration start time of the above-mentioned filtration process.

According to the embodiment described above, the required amount of sample water can be collected at a time at a specified water collection point in the surveyed water area W and stored in the storage container 21. Therefore, the required amount of sample water can be sampled quickly even in the surveyed water area W where there are rivers and ocean currents. Then, by discharging the collected sample water into the water area through a filtration device, fine biological fragments and excrement contained in the water (environment) of the surveyed water area W can be captured by the filtration device. In other words, it is possible to suitably collect environmental DNA samples required for analysis while adjusting for the long filtration time. Therefore, it is possible to obtain biological information in the surveyed water area W and perform appropriate environmental DNA evaluation. Moreover, instead of using the storage container 21 containing the sample water, the sample captured by the filtration device can be brought back to an analysis facility, etc., which suitably reduces the labor required for analysis.

In addition, a single filtration set is formed by combining the storage container 21 and the filtration device, and since the number of filtration sets required for the number of collections is provided, the required amount of sample water can be collected in the storage container 21 of each filtration set at each sampling time, while the environmental DNA samples required for analysis can be collected individually by each filtration device. This further reduces the labor involved in analyzing environmental DNA. In addition, since the number of filtration sets required for the number of collections is provided, the influence of the sample water collected previously can be eliminated. In addition, in the case of water quality that makes it difficult to filter a large amount of sample water, the amount that has been filtered can be understood by checking the amount of sample water (remaining amount) remaining in the storage container 21. Furthermore, by replacing the filtration set with a new one when collecting it, the environmental DNA samples required for analysis can be collected continuously.

In addition, at least the second filter 32 of the filtration device is installed in a temperature-controlled refrigerator room 35, which prevents deterioration of minute biological fragments and excrement captured by the filtration device. This makes it possible to obtain accurate biological information in the surveyed water area W. This allows for an appropriate evaluation of environmental DNA.

In addition, in the environmental DNA collection method of this embodiment, the collection device installation step installs an environmental DNA sample collection device M in the surveyed water area W, and the water collection step allows sample water to be collected at each sampling time and stored in the storage container 21 of each filtration set. In other words, the required amount of sample water collected at a certain specified water collection point and sampling time can be secured in the storage container 21. Therefore, the required amount of sample water can be quickly sampled even in an actual sea area with a current. Then, the filtration step allows the sample water in the storage container 21 to be drained through the filtration device, so that fine biological fragments and excrement contained in the water (environment) of the surveyed water area W can be captured by the filtration device. Then, the recovery step allows the filtration residue to be recovered as a sample for environmental DNA analysis, so that biological information in the surveyed water area W can be obtained easily and appropriate environmental DNA can be evaluated. Moreover, the filtration residue can be recovered on-site in the surveyed water area W, so that the labor required for analysis can be suitably reduced.

Next, another configuration of the environmental DNA sample collection device will be described with reference to Fig. 3. In Fig. 3, parts similar to those in the above embodiment are given the same reference numerals and duplicated explanations will be omitted. The environmental DNA sample collection device M1 is provided on the structure body 110 of the floating structure 100, as in the above embodiment.
As shown in Fig. 3, the environmental DNA sample collection device M1 includes a water sampling pump 10, a storage tank 21A as a storage container, a filtration device group 30A, and a filtration pump 50. The filtration device group 30A includes two filtration devices (two filtration sets) that share the storage tank 21A as a storage container. The filtration pump 50 is configured to directly suck the upstream side of the discharge pipe 17.
Although not shown, the environmental DNA sample collecting device M1 also includes the control unit 60 described in the above embodiment, a battery 70, and a solar panel (power generation equipment) 80 (see FIG. 2).
The discharge pipe 12 is connected to the top of the storage tank 21A via a first filter 31. In other words, the storage tank 21A is configured so that sample water from which larger particles and unnecessary substances have been removed by the first filter 31 flows in. An upstream suction port of the outlet pipe 19 is disposed via the first filter 31 at the lower inside portion of the storage tank 21A. The outlet pipe 19 extends toward the branch pipe 13 in the subsequent stage and is connected to the branch pipe 13. A flow meter 19b and a seventh opening/closing valve 19a for opening and closing the outlet pipe 19 are provided midway along the outlet pipe 19.

Outlet pipes 15, 15 are connected to both ends of the branch pipe 13. An eighth on-off valve 15A and a ninth on-off valve 15B for opening and closing the outlet pipe 15 are provided on the upstream and downstream sides of each outlet pipe 15. The downstream side of the ninth on-off valve 15B of each outlet pipe 15 is connected to a collecting drain pipe 16A via a first filter 31 and a second filter 32 in the refrigerator compartment 35 and a fifth on-off valve 16a in the subsequent stage.
A first three-way valve 13A is provided in the middle of the branch pipe 13. A common-washing pipe 13C is connected to the first three-way valve 13A. The first three-way valve 13A is a valve that switches between two states: a state in which the branch pipe 13 and the common-washing pipe 13C are blocked while communicating with the branch pipe 13, and a state in which the branch pipe 13 and the common-washing pipe 13C are communicated with each other. A tenth opening/closing valve 13B that opens and closes the common-washing pipe 13C is provided in the middle of the common-washing pipe 13C.
The downstream end of the common-washing pipe 13C is connected to the second three-way valve 16B. The second three-way valve 16B is provided in the middle of the collecting drain pipe 16A. The second three-way valve 16B is a valve that switches between two states: a state in which the collecting drain pipe 16A communicates with the common-washing pipe 13C and the state in which the collecting drain pipe 16A communicates with the common-washing pipe 13C while blocking communication between the collecting drain pipe 16A and the common-washing pipe 13C.
A discharge pipe 17 is connected to the collecting drain pipe 16A. A sixth opening/closing valve 17a and a filtration pump 50 are provided in the middle of the discharge pipe 17.

Next, a method for collecting environmental DNA samples using the environmental DNA sample collecting device M1 will be described in detail.
The method for collecting environmental DNA samples includes a collection device installation step, a water collection step, a filtration step, and a recovery step, as in the above embodiment. Before the water is collected, the water collection pump 10 and the filtration pump 50 are stopped, and each opening and closing valve is closed.

In the sampling device installation process, a floating structure 100 (see FIG. 1, the same applies below) is moved to the surveyed water area W, and the floating structure 100 is moored in the surveyed water area W in the same manner as in the above embodiment.
In the water sampling process, sample water is sampled and stored in the storage tank 21A. In this case, prior to the start of filtration, the control unit 60 operates the water sampling pump 10 to temporarily store the sampled seawater in the storage tank 21A. Thereafter, the seawater is drained from the storage tank 21A by manually operating the discharge cock of the drainage pipe (not shown), and the water sampling pipe 11, discharge pipe, and storage tank 21A are washed together. It is preferable to drain the water from the storage tank 21A at a position away from the water sampling port 11a. This drainage can also be performed automatically under the control of the control unit 60.
Thereafter, when the filtration start time arrives, the control unit 60 operates the water sampling pump 10 again. As a result, the collected sample water is stored in the storage tank 21A. At this time, larger particles and unwanted matter contained in the sample water are primarily filtered and removed by the first filter 31.

In the filtration step, the control unit 60 first opens the seventh on-off valve 19a of the discharge pipe 19 and operates the first three-way valve 13A of the branch pipe 13 to communicate the branch pipe 13 with the common-washing pipe 13C. The control unit 60 also opens the tenth on-off valve 13B of the common-washing pipe 13C and operates the second three-way valve 16B to communicate the common-washing pipe 13C with the collecting drain pipe 16A. The control unit 60 also opens the sixth on-off valve 17a and operates the filtration pump 50. As a result, the sample water in the storage tank 21A flows through the discharge pipe 19, the branch pipe 13, the common-washing pipe 13C, the collecting drain pipe 16A, and the discharge pipe 17, and these are common-washed.
Thereafter, the control unit 60 operates the first three-way valve 13A of the branch pipe 13 to connect the branch pipe 13 and block the connection between the branch pipe 13 and the co-washing pipe 13C so that the sample water flows into one of the filtration devices (the first filter 31 and the second filter 32). The control unit 60 also operates the second three-way valve 16B to connect the collecting drain pipe 16A and block the connection between the collecting drain pipe 16A and the co-washing pipe 13C. Furthermore, the control unit 60 opens the eighth on-off valve 15A, the ninth on-off valve 15B, and the fifth on-off valve 16a of the outlet pipe 15 leading to one of the filtration devices, and operates the filtration pump 50.
Then, the sample water flows from the outlet pipe 19 through the branch pipe 13 to one of the outlet pipes 15, and the sample water filtered by the first filter 31 is further filtered by the second filter 32. As a result, minute biological debris, excrement, and the like contained in the sample water are captured by the second filter 32.
The control unit 60 controls the filtration to stop after a preset time has elapsed after the start of filtration, for example, one hour or 30 minutes, or when measurement by the flow meter 19b detects that a predetermined amount of sample water has passed.
Then, the control unit 60 starts the water sampling process in the other filtration set, for example, two hours after the filtration start time of the above-mentioned filtration process.

In the environmental DNA sample collection device M1 having the other configuration described above, the required amount of sample water collected at a predetermined water collection point in the surveyed water area W at the sampling time can be secured in the storage tank 21A. Therefore, a predetermined amount of sample water can be quickly sampled even in an actual sea area with a current. Then, by discharging the sample water in the storage tank 21A into the water area through a filtration device, minute biological fragments and excrement contained in the water (environment) of the surveyed water area W can be captured by the second filter 32 of the filtration device. Therefore, it is possible to obtain biological information in the water area and perform appropriate environmental DNA evaluation. Moreover, since the sample captured by the filtration device can be brought back to an analysis facility, the labor required for analysis can be suitably reduced.
In the above embodiment, the first filter 31 is provided in both the storage tank 21A and the filtering device group 30, but this is not limited thereto, and it may be provided in either one of them.

Although an embodiment of the present invention has been described above, the present invention is not limited to the above embodiment, and each component can be appropriately modified without departing from the spirit of the present invention.
For example, in the above embodiment, the environmental DNA sample collection device M, M1 is provided on a floating structure 100, but this is not limited to this, and the environmental DNA sample collection device M, M1 may also be provided on an above-water structure 150 constructed on the water with supports 140 as shown in Figure 4.
Furthermore, as shown in FIG. 5, environmental DNA sample collection devices M, M1 may be provided on a ship 170 moored in the survey waters W by a wire 101, an anchor 102, and a buoy 160.

In addition, in the above embodiment, the second filter 32 is placed inside the refrigerator compartment 35 cooled by the cooling device 33, but this is not limited to this, and the second filter 32 may be placed inside the freezer compartment.
In addition, in the above embodiment, a filtration device consisting of a first filter 31 and a second filter 32 was shown, but this is not limited to this, and a third filter with a different mesh size may be placed upstream or downstream of the first filter 31, etc.
In addition, in the above embodiment, the environmental DNA sample collection devices M, M1 are installed on the ocean, but they may also be installed on land adjacent to the survey water area W.
Furthermore, the storage container 21 and storage tank 21A may be made of any material as long as they are capable of storing a predetermined amount of sample water so as not to affect the analysis of environmental DNA.

21 Storage container 21A Storage tank (storage container)
31 First filter (filtration device)
32 Second filter (filtration device)
33 Cooling device 35 Refrigerator 100 Floating structure M, M1 Environmental DNA sample collection device W Survey water area

Claims (4)

An environmental DNA sample collection device to be installed in an environmental DNA survey water area, comprising:
It is equipped with a sample water storage container , a filtration device , a wastewater storage container, and a filtration pump .
At the time of collection, a required amount of sample water is collected from the surveyed water area and stored in the storage container, the sample water stored in the storage container is supplied to the filtration device by the filtration pump, and the environmental DNA sample is collected by filtering the sample water with the filtration device;
The wastewater storage container is configured to temporarily store filtrate after being filtered by the filtration device,
The filtration pump sucks in gas from the internal space of the wastewater storage container, creating a negative pressure state inside the wastewater storage container, thereby promoting the drainage of filtered water, and the sample water stored in the storage container is continuously supplied to the filtration device, characterized in that the environmental DNA sample collection device. The storage container and the filtration device are combined to form one filtration set,
2. The environmental DNA sample collection device according to claim 1, wherein the filtration sets are provided in the number required for the number of collections. The environmental DNA sample collection device according to claim 1 or 2, characterized in that at least the filtration device is installed in a temperature-controlled environment. 4. A method for sampling environmental DNA by installing the environmental DNA sampling device according to claim 1 in an environmental DNA survey water area, comprising:
a sampling device installation process for installing an environmental DNA sampling device in a survey water area, the environmental DNA sampling device having a number of filtration sets, each of which is constituted by combining the storage container and the filtration device, corresponding to a number of preset sampling times;
A water sampling step of collecting and storing sample water in the storage container of the corresponding filtration set at the sampling time;
a filtering step of supplying sample water from the storage container to the filtering device, filtering the sample water, and discharging the filtered water;
a recovery step of recovering the filtration residue as a sample for environmental DNA analysis;
A method for collecting environmental DNA samples, comprising:

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