Environmental nucleic acid monitoring method

Abstract

To simplify a sampling step of organism-derived nucleic acid to make a larger scale or a wider range of environmental nucleic acid monitoring easy.SOLUTION: In an environmental nucleic acid monitoring method for monitoring an organism-derived nucleic acid present in the environment, environmental nucleic acid-containing material which contains organism-derived nucleic acid present in the environment and is accumulated in a vehicle which travelled in the environment is recovered from the vehicle, nucleic acid is extracted from the recovered environmental nucleic acid-containing material, and the extracted nucleic acid is analyzed to acquire environmental organism data regarding organisms present in the environment.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to methods for environmental nucleic acid monitoring. [Background technology]

[0002] Examples of methods for investigating or monitoring biological species present in the environment include a method of recovering biological nucleic acids from the environment, analyzing the recovered nucleic acids, and identifying the biological species from which the nucleic acids originate. Biological nucleic acids are nucleic acids that contain genetic information of an organism and are released from an organism into the environment, and are included in nucleic acids present in the environment, such as environmental DNA. As a method for recovering and analyzing such biological nucleic acids from the environment, for example, a method is known in which a water sample (environmental water sample) collected from a body of water such as a river is subjected to environmental DNA analysis (see, for example, Patent Document 1 and Patent Document 2). In addition, as a method for detecting terrestrial mammals, a method is known in which soil is collected near a camera (for example, soil is sampled at a depth of 2 cm every 2 m) in combination with the results of image analysis from a fixed camera, and environmental DNA analysis is performed (see, for example, Non-Patent Document 1), and a method is known in which air is collected at three locations in a zoo, and DNA is extracted from the collected air and analyzed (see, for example, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-99376 A [Patent Document 2] JP 2020-92645 A [Non-patent literature]

[0004] [Non-Patent Document 1] Kevin Leempoel et al. A comparison of eDNA to camera trapping for assessment of terrestrial mammal diversity. Proceedings of the Royal Society B. 2020 Jan 15, vol. 287(1918) [Non-Patent Document 2] Christina Lynggaard et al. Airborne environmental DNA for terrestrial vertebrate community monitoring. Current Biology 32, 701-707, February 7, 2022 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, in order to recover biological nucleic acids from the environment, a dedicated active sampling process is generally required. Specifically, sampling processes such as collecting environmental water samples from water areas designated as targets for recovering biological nucleic acids, collecting soil samples from specific locations on land areas designated as targets for recovering biological nucleic acids, or collecting air samples from specific locations on land areas designated as targets for recovering biological nucleic acids are required. In recent years, surveys or monitoring of biological species present in the environment are not only conducted for academic purposes, but are also becoming common methods with the participation of government agencies and private companies. Therefore, in order to realize the above surveys and monitoring on a large scale and over a wide area, there has been a demand for a technology that simplifies the sampling process of biological nucleic acids and enables biological nucleic acids to be sampled easily from a wider area. [Means for solving the problem]

[0006] The present disclosure can be realized in the following forms. (1) According to one aspect of the present disclosure, there is provided an environmental nucleic acid monitoring method for monitoring biological nucleic acids present in an environment. This environmental nucleic acid monitoring method involves recovering environmental nucleic acid-containing material from a vehicle that has traveled through an environment, which contains biological nucleic acids present in the environment and has accumulated in the vehicle, extracting nucleic acids from the recovered environmental nucleic acid-containing material, and analyzing the extracted nucleic acids to obtain environmental biological data regarding organisms present in the environment. According to this form of environmental nucleic acid monitoring method, biological nucleic acids can be sampled by taking advantage of the fact that environmental nucleic acid-containing materials adhere to a vehicle as the vehicle travels through the environment, making it easier to conduct large-scale and wide-area surveys and monitoring of environmental organisms, and simplifying the process of sampling biological nucleic acids, making it possible to easily sample biological nucleic acids from a wider area. (2) In the environmental nucleic acid monitoring method of the above aspect, the environmental biological data may include data containing information on at least one taxonomic rank of the organism from which the nucleic acid is derived, which is identified by analyzing the nucleic acid extracted from the environmental nucleic acid-containing material. With this configuration, biological nucleic acid can be monitored based on at least one taxonomic rank of the organism from which the nucleic acid in the recovered environmental nucleic acid-containing material is derived. (3) In the environmental nucleic acid monitoring method of the above aspect, the environmental biological data and geographical data including at least one of information regarding the vehicle's travel route and map data may be used to generate biodiversity information including information regarding the geographical distribution of organisms. With this configuration, biological nucleic acids can be monitored in association with the geographical data. The present disclosure can be realized in various forms other than those described above, for example, a method for recovering nucleic acid derived from a living organism, a method for extracting nucleic acid derived from a living organism, a method for analyzing nucleic acid derived from a living organism, etc. [Brief description of the drawings]

[0007] [Figure 1] 1 is a flow chart showing a method for environmental nucleic acid monitoring. [Diagram 2] FIG. 1 is an explanatory diagram showing locations where environmental nucleic acid-containing substances adhere in a wheel house. [Diagram 3] 1 is a flow chart showing an example of a method for extracting biological nucleic acid. [Figure 4] 1 is a flowchart showing another example of a method for extracting biological nucleic acid. [Diagram 5] FIG. 1 is an explanatory diagram conceptually illustrating an example of how biodiversity information is collectively displayed. [Figure 6] FIG. 1 is an explanatory diagram showing the results of analyzing environmental DNA samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] A. Overview of Environmental Nucleic Acid Monitoring Methods: FIG. 1 is a flow chart showing an environmental nucleic acid monitoring method according to an embodiment. The "environmental nucleic acid" monitored in this embodiment is a nucleic acid present in the environment, including nucleic acid derived from organisms present in the environment. Here, the "organism-derived nucleic acid" is a substance that constitutes an organism as a nucleic acid containing genetic information of the organism. In this specification, the term "organism" refers to a general term for organisms including animals, fungi, plants, protozoa, archaea, bacteria, etc., and further includes viruses. The organism-derived nucleic acid to be monitored by the environmental nucleic acid monitoring method of this embodiment can be deoxyribonucleic acid (DNA) and may include ribonucleic acid (RNA). In the environmental nucleic acid monitoring method of this embodiment, in order to collect organism-derived nucleic acid mainly from land areas in the environment, a vehicle is used to collect environmental nucleic acid-containing material containing organism-derived nucleic acid. The environmental nucleic acid-containing material includes cells and tissue pieces peeled off from various organisms, excrement-derived material, and material (fragments, etc.) derived from the corpses of organisms. The environmental nucleic acid-containing material can be present in various places on land, such as on the surface of the earth, on the ground, and in the air. Environmental nucleic acid-containing materials present on the earth's surface include biological nucleic acids derived from various biological species that exist above the earth's surface (on the ground, on the ground, in the air, etc.).

[0009] The researcher who monitors environmental nucleic acids first collects environmental nucleic acid-containing materials from a vehicle that has traveled in the environment (step T100). This allows the collection of environmental nucleic acid-containing materials from the places where the vehicle has traveled. When the vehicle travels, the environmental nucleic acid-containing materials that are mainly present on the ground surface at the places where the vehicle has traveled adhere to the vehicle and accumulate. In step T100, the environmental nucleic acid-containing materials that have accumulated on the vehicle while traveling are collected. Note that, from the viewpoint of associating the collection locations of environmental nucleic acids with the analysis results of environmental nucleic acids, it is desirable to use vehicles with a fixed travel route for the vehicle used for environmental nucleic acid monitoring. For example, the vehicle may be a bus that travels along a predetermined route, or a motorsport vehicle that travels within a specific area such as a rally.

[0010] The target location for recovering accumulated environmental nucleic acid-containing substances in a vehicle is not particularly limited, as long as it is a location where environmental nucleic acid-containing substances can accumulate during driving and the accumulated environmental nucleic acid-containing substances can be recovered after driving, and can be the surface of the vehicle body or any location through which outside air flows during driving. If, for example, vehicle tires, the inside of the wheelhouse where the tires are placed, radiator fins, engine air filters, air conditioner filters, etc. are used as the target location for recovering environmental nucleic acid-containing substances, it becomes possible to recover environmental nucleic acid-containing substances more efficiently.

[0011] Environmental nucleic acid-containing substances that were mainly present on the ground surface accumulate on the surface of the tire and on the outermost surface of the inside of the wheelhouse as the vehicle travels. For example, when the tire rotates while in contact with the ground surface during vehicle travel, environmental nucleic acid-containing substances from the ground surface adhere to the surface. Furthermore, environmental nucleic acid-containing substances that are kicked up by the tire during vehicle travel adhere to the inside of the wheelhouse. Here, the outermost surface of the wheelhouse to which the environmental nucleic acid-containing substances adhere refers to the surface of the fender liner that faces the tire when a fender liner is attached to the wheelhouse, for example.

[0012] Fig. 2 is an explanatory diagram showing, with hatching, adhesion sites 10 where environmental nucleic acid-containing substances mainly adhere in wheel houses 22 of tires 24 equipped on a vehicle 20. In Fig. 2, the direction of rotation of tires 24 when the vehicle 20 moves forward is indicated by an arrow. Fig. 2 shows the external appearance of the front part of the left side surface of the vehicle 20, but any of the four wheel houses 22 equipped on the vehicle 20 may be the target for collection of environmental nucleic acid-containing substances.

[0013] Furthermore, environmental nucleic acid-containing substances that have been picked up from the ground by the tires and mixed into the air or that have been suspended in the air adhere to the surfaces of the radiator fins of the radiator mounted on the vehicle when the vehicle is moving and the wind passes through the radiator.Similarly, environmental nucleic acid-containing substances that have been picked up from the ground by the tires and mixed into the air or that have been suspended in the air adhere to the surfaces of engine air filters and air conditioner filters when the outside air passes through these filters.

[0014] In step T100, the environmental nucleic acid-containing material that has adhered to and accumulated at the target site of collection of the environmental nucleic acid-containing material is collected. Specifically, for example, the surface of the target site of collection may be wiped with a collection member to collect the environmental nucleic acid-containing material. The collection member used may be, for example, a cotton bud-like member (swab-like member) or a cloth-like member.

[0015] After collecting the environmental nucleic acid-containing material from the vehicle in step T100, the investigator extracts nucleic acid from the collected environmental nucleic acid-containing material (step T110).

[0016] Fig. 3 is a flow chart showing an example of a method for extracting biological nucleic acid. Fig. 3 shows a case where nucleic acid is extracted directly from a recovery member when a relatively small recovery member such as the swab-shaped member described above is used. When extracting biological nucleic acid, first, a recovery member having environmental nucleic acid-containing matter attached thereto is prepared (step T200). This recovery member is the one from which environmental nucleic acid-containing matter was recovered from a recovery target location in a vehicle in step T100 in Fig. 1.

[0017] The recovery member is then crushed and suspended in a liquid (step T210). The liquid used here may be a lysis buffer that dissolves cells and tissues. The recovery member may be crushed by adding, for example, ceramic beads or the like to the liquid in addition to the recovery member and stirring. By crushing the recovery member in this manner, the environmental nucleic acid-containing material attached to the recovery member is peeled off from the recovery member, and the biologically derived nucleic acid in the environmental nucleic acid-containing material is eluted into the suspension in which the recovery member is crushed.

[0018] Thereafter, the biological nucleic acid is extracted from the suspension in which the biological nucleic acid has been eluted (step T220). The biological nucleic acid may be extracted, for example, by centrifuging the suspension, recovering the liquid in which the biological nucleic acid has been dissolved as a supernatant, selecting an appropriate column to remove impurities, and then purifying the biological nucleic acid using a column that adsorbs nucleic acid. Examples of the impurities include substances that inhibit the reaction included in the PCR step described below. The steps of nucleic acid extraction shown as steps T210 and T220 are known methods, and for example, a commercially available soil DNA extraction kit may be used. If such a method is used, nucleic acid extraction can be performed directly from the recovery member, and the steps related to nucleic acid extraction can be simplified.

[0019] Fig. 4 is a flow chart showing another example of a method for extracting biological nucleic acid. Fig. 4 shows a case where a relatively large recovery member such as a cloth member is used to recover an environmental nucleic acid-containing material from a recovery target location on a vehicle, and an operation for concentrating the environmental nucleic acid-containing material attached to the recovery member is performed prior to nucleic acid extraction. When extracting biological nucleic acid, first, a recovery member to which the environmental nucleic acid-containing material is attached is prepared (step T300), similar to step T200.

[0020] Thereafter, the prepared recovery member is washed in a liquid (step T310). The liquid used here may be a nuclease-free buffer or nuclease-free water that does not contain nuclease decomposition enzymes. By washing the recovery member in such a liquid, the environmental nucleic acid-containing material is peeled off from the recovery member and transferred into the liquid, forming a suspension. Thereafter, the suspension containing the environmental nucleic acid-containing material is filtered to obtain a residue containing the environmental nucleic acid-containing material, and the environmental nucleic acid-containing material is concentrated on the filter (step T320). The pore size of the filter used here may be appropriately selected so that the environmental nucleic acid-containing material contained in the material attached to the recovery member can be efficiently recovered. The pore size of the filter for concentrating the environmental nucleic acid-containing material is, for example, preferably 0.22 to 10 μm, more preferably 0.22 to 5 μm. During filtration, the filtration efficiency may be increased by, for example, applying negative pressure using a vacuum pump or applying pressure using a pump.

[0021] The substances attached to the recovery member may include unnecessary substances that are difficult to use as targets for extracting biological nucleic acids. Therefore, in order to increase the efficiency of concentrating the environmental nucleic acid-containing material by the above-mentioned filtration and the efficiency of the subsequent nucleic acid extraction, the unnecessary substances may be removed prior to the filtration for concentrating the environmental nucleic acid-containing material. In order to remove the unnecessary substances, the suspension may be filtered prior to concentrating the environmental nucleic acid-containing material using a filter (e.g., a pore size of about 100 μm) that is coarser than the filter used for concentrating the environmental nucleic acid-containing material. The obtained filtrate may then be subjected to filtration for concentrating the environmental nucleic acid-containing material. Examples of the unnecessary substances include relatively large plant fragments such as leaves, dead bodies of organisms such as insects, stones, etc.

[0022] When the filter containing concentrated environmental nucleic acid is obtained in step T320, the filter is then crushed and suspended in a liquid (step T330). This step T330 is the same as step T210 in FIG. 3. As a result, the environmental nucleic acid-containing material concentrated on the filter is peeled off from the filter, and the biological nucleic acid in the environmental nucleic acid-containing material is eluted into the suspension in which the filter is crushed. Then, the biological nucleic acid is extracted from the suspension in which the biological nucleic acid is eluted (step T340). Step T340 is the same as step T220 in FIG. 3. If such a method is used, even if the amount of environmental nucleic acid-containing material attached to the collection target portion of the vehicle is relatively small and the environmental nucleic acid-containing material is attached over a wide area of ​​the collection target portion, nucleic acid can be efficiently extracted.

[0023] Returning to FIG. 1, when nucleic acid is extracted from the environmental nucleic acid-containing material in step T110, the researcher analyzes the extracted nucleic acid to obtain environmental biological data (step T120). Nucleic acid analysis can be performed by various known methods. For example, species-specific analysis such as the determination of the presence or absence of a specific biological species or the quantification of the amount of the species by quantitative PCR (qPCR) can be performed. In this case, for example, by focusing on a specific biological species group such as birds or mammals and performing PCR by selecting appropriate primers, it is possible to narrow down the analysis to the specific biological species group. In addition, by selecting appropriate primers and performing PCR, it is also possible to identify a wider range of biological species at once. In addition, a comprehensive analysis for identifying biological species may be performed by comparing the base sequence obtained using a next-generation sequencer or the like with a database, or a haplotype analysis may be performed within the same species. In this way, information on organisms that inhabit or stay in the area where the environmental nucleic acid-containing material was collected (the route of the vehicle used to collect the environmental nucleic acid-containing material) can be obtained as environmental biological data.

[0024] For example, when a comprehensive analysis is performed to identify biological species by comparing the base sequence obtained using a next-generation sequencer or the like with a database as described above, data containing information on at least one of the taxonomic ranks of the organisms from which the nucleic acids contained in the environmental nucleic acid content originate can be obtained as environmental biological data. The "taxonomic rank of organisms" refers to ranks such as kingdom, phylum, class, order, family, genus, and species. By comparing the base sequence of nucleic acid obtained from the environment with the nucleic acid sequence registered in various databases, such as the National Center for Biotechnology Information (NCBI) in the United States and other public databases and self-created databases, the classification of the organism from which the nucleic acid is derived can be identified by any taxonomic rank suited to the purpose of the experiment.

[0025] B. Biodiversity information generation: When performing the environmental nucleic acid monitoring method of this embodiment, after the environmental biological data is acquired in step T120, biodiversity information including information on geographical biological distribution may be generated using the acquired environmental biological data and geographical data including at least one of information on the driving route of the vehicle used to collect the environmental nucleic acid-containing material and map data. Specifically, for example, when the vehicle used to collect the environmental nucleic acid-containing material is a bus, the above geographical data may include information indicating the driving route such as the route name or system name of the bus used, the name of the area including the driving route, or information for displaying the driving route superimposed on the map. In addition, when the vehicle used to collect the environmental nucleic acid-containing material is a rally vehicle, the above geographical data may include the name of the competition section in which the used vehicle ran, the name of the area including the competition section, or information for displaying the driving route as the competition section superimposed on the map. In addition, including the case where a vehicle other than the above-mentioned vehicle with a strictly defined driving route is used, the geographical data may include, for example, vehicle movement data that can be acquired from a digital tachometer, etc., specifically, position information by GPS, and information indicating the vehicle's driving distance corresponding to the sampling range. If the operation of acquiring environmental biological data as described above is repeated at regular intervals, for example each season, and the acquired environmental biological data is associated (integrated) with geographical data to generate biodiversity information, it is possible to monitor changes over time in the species of biological species inhabiting or residing in the target area.

[0026] FIG. 5 is an explanatory diagram conceptually illustrating an example of a state in which the biodiversity information generated as described above is displayed together. In FIG. 5, the geographical data is composed of the route name of the bus, which is the vehicle used to collect the environmental nucleic acid-containing material. In addition, the environmental biological data shown in FIG. 5 is data on birds and is composed of the species names of birds from which the nucleic acids contained in the environmental nucleic acid-containing material are derived. In FIG. 5, Bird A, Bird B, Bird C, etc. indicate the species names of the detected birds. In addition, FIG. 5 illustrates an example of the results of environmental nucleic acid monitoring performed by season in a specific year, such as January, April, July, and October of 2023.

[0027] The environmental biological data to be associated with the geographical data may be data including the names of all detected biological species, or may be data including the names of some of the detected biological species. Data including the names of some of the detected biological species may be, for example, data consisting of the names of biological species detected each time when multiple measurements are performed independently at a specific time, or data consisting of the names of biological species contained at a certain rate or more when quantitative analysis is performed. The content of the environmental biological data and the type of geographical data used when combining the environmental biological data and the geographical data may be appropriately set depending on the purpose of monitoring environmental nucleic acids, etc.

[0028] According to the environmental nucleic acid monitoring method of the present embodiment configured as described above, environmental nucleic acid-containing materials are collected from vehicles that have traveled in the environment, nucleic acids are extracted from the collected environmental nucleic acid-containing materials, and the extracted nucleic acids are analyzed to obtain environmental biological data. In this way, biological nucleic acids can be sampled by utilizing the fact that environmental nucleic acid-containing materials adhere to the vehicle as the vehicle travels, which makes it easier to survey and monitor environmental organisms on a large scale and over a wide area, and simplifies the process of sampling biological nucleic acids, making it possible to easily sample biological nucleic acids from a wider area.

[0029] In this case, if the target site for collection of environmental nucleic acid-containing substances is a vehicle's tire, the inside of a wheel house, a radiator fin, an engine air filter, an air conditioner filter, etc., it is possible to simply and efficiently collect environmental nucleic acid-containing substances by using the parts that the vehicle originally has, without providing any special parts for sampling. In addition, if a bus that runs on a predetermined route or a motorsport vehicle that runs in a specific area such as a rally is used as a vehicle used for collection of environmental nucleic acid-containing substances, there is no need to run the vehicle specially for sampling biological nucleic acid, and sampling can be performed more simply. When using such a vehicle, a vehicle that runs in a desired area can be selected for each area for the purpose of environmental nucleic acid monitoring or for comparison of environmental nucleic acid, and used as the target for collection of environmental nucleic acid-containing substances.

[0030] C. Other embodiments: In the above-described embodiment, the vehicle used as the subject for collecting the environmental nucleic acid-containing substance is used as is, and the environmental nucleic acid-containing substance is collected from the parts originally equipped in the vehicle, but a different configuration is also possible. For example, a collection member for adhering the environmental nucleic acid-containing substance may be attached in advance to a portion of the vehicle where the environmental nucleic acid-containing substance is likely to accumulate, specifically, the tire surface, the inside of the wheel house, the surface of the radiator fin, etc., and after the collection member is collected from the vehicle, biological nucleic acid is extracted from the collection member. In this case, the same effect of simplifying the sampling process of biological nucleic acid can be obtained. EXAMPLES

[0031] According to the environmental nucleic acid monitoring method of the present disclosure, biological nucleic acids were collected from the environment, and biological species were identified from the obtained biological nucleic acids. Specifically, two 20 km sections in Aichi Prefecture were set as collection target areas. Then, in April 2021 and June 2021, biological nucleic acids were collected in each collection target area. Here, a vehicle was driven in each of the above-mentioned 20 km sections, and environmental nucleic acid-containing substances were attached to the tires of the vehicle. Thereafter, the environmental nucleic acid-containing substances attached to the tires were collected by pressing a collection member (swab) made of chemical fiber against the tire, and biological nucleic acids were extracted from the collection member according to the method of FIG. 3. In step T110, DNA was extracted from the collection member using a commercially available DNA extraction kit for soil samples (NucleoSpin Soil, Takara Bio Inc.) (NucleoSpin is a registered trademark).

[0032] The nucleic acid extract obtained using the above kit was used as an environmental DNA sample from the target collection area, and the 12SrRNA sequence was amplified by PCR using a specific primer (an existing avian universal primer). The Kapa Hifi PCR Kit (KAPABIOSYSTEMS) was used for amplification, and PCR was repeated four times. Furthermore, PCR was performed to add an index for sequence analysis, and the PCR product was purified with beads. After that, the sequence was obtained using a next-generation sequencer. The sequencer used was Illumina iSeq100 (Illumina, Inc.) (iSeq is a registered trademark).

[0033] FIG. 6 is an explanatory diagram showing the analysis results of the above-mentioned environmental DNA sample. For the obtained sequences, the biological species was extracted using MiFish Pipeline, a pipeline for DNA analysis, and further, a BLAST homology search was performed on the obtained sequences to attempt to identify the biological species. Among them, only biological species with a homology of 97% or more with the database are listed in FIG. 6. FIG. 6 shows the results of identifying biological species at the genus level. As shown in FIG. 6, it was confirmed that a wide variety of terrestrial birds live or have stayed there by the method of recovering biological nucleic acids of the present disclosure. In addition, similar results were obtained, showing that more bird species live in spring (April) than in summer (June) in two relatively nearby recovery target areas.

[0034] The present disclosure is not limited to the above-mentioned embodiments, etc., and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features in the embodiments corresponding to the technical features in each form described in the Summary of the Invention column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted. [Explanation of symbols]

[0035] 10…Attachment location 20…Vehicle 22…Wheelhouse 24…Tire

Claims

1. An environmental nucleic acid monitoring method for monitoring biological nucleic acids present in an environment, comprising the steps of: recovering environmental nucleic acid-containing material from the vehicle that has traveled through the environment, the environmental nucleic acid-containing material including nucleic acid of biological origin present in the environment and accumulated in the vehicle; Extracting nucleic acids from the collected environmental nucleic acid-containing material; The extracted nucleic acid is analyzed to obtain environmental biological data regarding organisms present in the environment. Environmental nucleic acid monitoring methods.

2. The method for environmental nucleic acid monitoring according to claim 1, As the environmental organism data, data including information on at least one taxonomic rank of an organism from which the nucleic acid is derived, which is identified by analyzing the nucleic acid extracted from the environmental nucleic acid-containing material, is obtained. Environmental nucleic acid monitoring methods.

3. The method for environmental nucleic acid monitoring according to claim 1 or 2, further comprising: Using the environmental biological data and geographical data including at least one of information regarding the vehicle's travel route and map data, biodiversity information including information regarding geographical biological distribution is generated. Environmental nucleic acid monitoring methods.

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