Bio-derived nucleic acid recovery attachment

Abstract

To recover nucleic acids derived from terrestrial organisms over an expansive target range.SOLUTION: A biological nucleic acid recovery attachment includes a mounting part for mounting the biological nucleic acid recovery attachment to the inside of a wheelhouse of a tire of a vehicle, and a collecting part that entraps and retains biological nucleic acids, flung up by the tire during the vehicle's travel.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a bio-derived nucleic acid recovery attachment for attaching to a vehicle to recover bio-derived nucleic acids.

Background Art

[0002] As a method for investigating or monitoring biological species present in the environment, there is a method of recovering bio-derived nucleic acids from the environment, analyzing the recovered nucleic acids, and identifying the biological species from which the nucleic acids are derived. Bio-derived nucleic acids are nucleic acids that contain the genetic information of organisms and are released from organisms 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 bio-derived nucleic acids from the environment, for example, a method of subjecting a water sample (environmental water sample) collected from a water area such as a river to environmental DNA analysis is known (see, for example, Patent Document 1 and Patent Document 2). In addition, as a method for detecting terrestrial mammals, in combination with the video analysis results of a fixed-point camera, soil is collected near the camera (for example, soil is sampled at a depth of 2 cm every 2 m), and a method of performing environmental DNA analysis is known (see, for example, Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] However, when analyzing water samples, only nucleic acids from fish, waterfowl, or animals that inhabit the water body from which the sample was collected can be recovered. Therefore, there has been a desire to recover and analyze nucleic acids derived from terrestrial organisms that live far from aquatic environments. In particular, since nucleic acids are easily degraded in water, recovering nucleic acids from land, where they exist in a relatively less degradable dry state, is expected to enable comprehensive surveying and monitoring of existing biological species. As a method for recovering nucleic acids derived from terrestrial organisms, when soil is collected as the target of analysis, as described in Non-Patent Literature 1, it is difficult to conduct sampling over a wide area. Therefore, there has been a need for a technology that can recover nucleic acids derived from terrestrial organisms over a wide area. [Means for solving the problem]

[0006] This disclosure can be implemented in the following forms: (1) According to one embodiment of the present disclosure, a biological nucleic acid recovery attachment is provided. The biological nucleic acid recovery attachment comprises an attachment portion for attaching the biological nucleic acid recovery attachment to the inside of the wheel well of a tire of a vehicle, and a collection portion for taking in and holding biological nucleic acids that are thrown up by the tire as the vehicle is driven. This type of bio-based nucleic acid recovery attachment allows for the accumulation and recovery of bio-based nucleic acids within the attachment while the vehicle is in motion by installing it inside the vehicle's wheel well. Therefore, by driving a vehicle equipped with the bio-based nucleic acid recovery attachment in a designated recovery area, bio-based nucleic acids can be efficiently recovered from that area. More specifically, surface sediments containing bio-based nucleic acids can be efficiently recovered. By extracting bio-based nucleic acids from the recovered surface sediments, comprehensive analyses, such as identifying the species of organisms present in the recovery area, can be efficiently performed. Here, since the bio-based nucleic acids recovered by the bio-based nucleic acid recovery attachment may include not only nucleic acids derived from organisms living on the surface but also bio-based nucleic acids released into the atmosphere, it becomes possible to recover bio-based nucleic acids from a wider range of organisms that inhabit or reside in the recovery area. (2) In the above-described bio-derived nucleic acid recovery attachment, the collection portion may include a wall portion attached to the wheel house along the inner wall surface of the wheel house, a holding space forming portion located below the wall portion when the bio-derived nucleic acid recovery attachment is attached to the inside of the wheel house, and forming a holding space for holding the bio-derived nucleic acid between itself and the wall portion, and an opening provided at a position facing the tire, which connects the holding space with the outside of the bio-derived nucleic acid recovery attachment. With such a configuration, by attaching the bio-derived nucleic acid recovery attachment to the inside of the wheel house of the vehicle, it becomes possible to accumulate and recover bio-derived nucleic acids in the holding space while the vehicle is running. (3) The above-described bio-derived nucleic acid recovery attachment may further include a mesh portion covering the opening. With this configuration, surface sediments containing bio-derived nucleic acids can be recovered while excluding those with a pore diameter larger than that of the mesh portion. Therefore, by appropriately setting the pore diameter of the mesh portion, for example, surface sediments of a size considered efficient for the extraction of bio-derived nucleic acids can be recovered. (4) In the above-described bio-derived nucleic acid recovery attachment, the pore diameter of the mesh portion may be 1 cm or less. With such a configuration, surface sediments that are thought to contain a relatively high amount of bio-derived nucleic acids can be recovered efficiently. (5) In the above-described bio-derived nucleic acid recovery attachment, the collection section may be provided with a porous body having pores formed therein in which the bio-derived nucleic acid is taken in. With such a configuration, the bio-derived nucleic acid can be recovered by holding it in the pores of the porous body. (6) The above-described bio-derived nucleic acid recovery attachment may further include a frame portion having a space for housing the porous body formed inside and the mounting portion provided therein. With this configuration, bio-derived nucleic acids can be recovered by holding them in the pores of the porous body which is attached to the inside of the wheel well of the vehicle via the frame portion. (7) In the above-described form of the bio-derived nucleic acid recovery attachment, the mounting portion may be provided with mounting holes at positions corresponding to the placement positions of mounting members for attaching the fender liner to the wheel well, into which the mounting members are fitted. With this configuration, when attaching the fender liner to the wheel well, the fender liner and the bio-derived nucleic acid recovery attachment can be attached to the wheel well simultaneously using mounting members common to the fender liner. Therefore, the increase in the number of parts for attaching the bio-derived nucleic acid recovery attachment can be suppressed. This disclosure can be implemented in various forms other than those described above, for example, in the form of a vehicle equipped with a bio-derived nucleic acid recovery attachment, a method for recovering surface sediments, or a method for recovering bio-derived nucleic acids. [Brief explanation of the drawing]

[0007] [Figure 1] An explanatory diagram showing the schematic configuration of a vehicle equipped with a bio-derived nucleic acid recovery attachment. [Figure 2]An explanatory diagram showing the schematic configuration of a vehicle equipped with a bio-derived nucleic acid recovery attachment. [Figure 3] An explanatory diagram showing the appearance of the biological nucleic acid recovery attachment of the first embodiment. [Figure 4] An explanatory diagram showing how to attach the bio-derived nucleic acid recovery attachment. [Figure 5] An explanatory diagram showing the results of DNA sample analysis from the inside of a wheel well. [Figure 6] An explanatory diagram showing the results of DNA sample analysis from the tire surface. [Figure 7] An explanatory diagram showing the configuration of the biological nucleic acid recovery attachment of the second embodiment. [Figure 8] An explanatory diagram showing the configuration of the biological nucleic acid recovery attachment of the third embodiment. [Modes for carrying out the invention]

[0008] A. First Embodiment: (A-1) Configuration of the bio-based nucleic acid recovery attachment and operation of bio-based nucleic acid recovery: Figures 1 and 2 are explanatory diagrams illustrating the schematic configuration of a vehicle 20 equipped with a biological nucleic acid recovery attachment 10 according to the first embodiment. Figure 1 shows the external appearance of the front portion of the left side of the vehicle 20, and Figure 2 shows the arrangement of the biological nucleic acid recovery attachment 10 when the vehicle 20 is viewed from the front right. Figures 1 and 2, and each of the figures described later, show mutually orthogonal XYZ axes to specify the direction. The X, Y, and Z axes shown in each figure represent the same direction. In this specification, the X axis indicates the front-rear direction of the vehicle, the Y axis indicates the width direction of the vehicle, and the Z axis indicates the up-down direction of the vehicle.

[0009] As shown in Figure 1, the bio-derived nucleic acid recovery attachment 10 of this embodiment is used by being attached to the rear (-X direction) portion of the wheel well 22 of the tire 24 of the vehicle 20. In Figure 1, the direction of rotation of the tire 24 when the vehicle 20 is moving forward is indicated by an arrow. As described above, the bio-derived nucleic acid recovery attachment 10 attached to the rear side of the vehicle inside the wheel well 22 recovers bio-derived nucleic acids that are kicked up by the tire 24 when the vehicle 20 is moving. Specifically, it recovers surface sediments containing bio-derived nucleic acids that are kicked up by the tire 24. In this embodiment, the bio-derived nucleic acid recovery attachment 10 is attached to the inside of the wheel well 22 of all four tires 24 of the vehicle 20, but the bio-derived nucleic acid recovery attachment 10 may be attached to the inside of any one or more of the four wheel wells 22.

[0010] Figure 3 is an explanatory diagram showing the external appearance of the biological nucleic acid recovery attachment 10. The biological nucleic acid recovery attachment 10 comprises a collection section 12 and an attachment section 17.

[0011] The collection unit 12 collects and holds surface sediments containing biologically derived nucleic acids that are kicked up by the tires 24 of the vehicle 20 as the vehicle moves. The collection unit 12 in this embodiment comprises a wall portion 13, a holding space forming portion 14, and a pair of side portions 15.

[0012] The wall surface portion 13 is a rectangular plate-like member, bent into a shape along the inner wall surface of the wheel house 22, and attached to the inner wall surface of the wheel house 22. The holding space forming portion 14 is a rectangular plate-like member having the same width as the wall surface portion 13 and a shorter length in the height direction than the wall surface portion 13, provided continuously from the lower end of the wall surface portion 13, and has a shape bent upward from the lower end of the wall surface portion 13. The upper side of the holding space forming portion 14 is spaced apart from the wall surface portion 13. The distance between the upper side of the holding space forming portion 14 and the wall surface portion 13 is shown as distance D in FIG. 2. The larger the distance D, the larger the amount of surface sediment that can be held in the biological origin nucleic acid recovery attachment 10, but the size of the distance D may be appropriately set according to the distance between the inner wall surface of the wheel house 22 and the tire 24. The size of the distance D may be, for example, about 1 cm.

[0013] The side surface portion 15 is a plate-like member formed to project forward (+X-axis direction) of the vehicle from each of the sides (sides extending along the Z-axis direction) at both ends in the width direction of the wall surface portion 13, and has a side γ connecting the end A of the upper side α of the wall surface portion 13 and the end B of the upper side β of the holding space forming portion 14 as a part of the outer periphery. In the collection portion 12, a holding space 16 for holding surface sediment is formed as a space surrounded by the wall surface portion 13, the holding space forming portion 14, and the side surface portion 15. And an opening 18 is formed surrounded by the upper side α of the wall surface portion 13, the upper side β of the holding space forming portion 14, and the side γ connecting the above-described end A and end B in the side surface portion 15. The opening 18 is provided at a position facing the tire 24 and communicates the holding space 16 with the outside of the biological origin nucleic acid recovery attachment 10. When the tire 24 kicks up the surface sediment, the kicked-up surface sediment enters the holding space 16 from the opening 18 and is held in the holding space 16.

[0014] Note that the holding space forming portion 14 may have a shape different from that described above, and when the biological origin nucleic acid collection attachment 10 is attached inside the wheel house 22, it may be located on the lower side of the wall surface portion 13, and it is sufficient if a holding space 16 for holding the biological origin nucleic acid can be formed between the holding space 16 and the wall surface portion 13. Also, the side surface portion 15 is not essential, but in order to suppress the dropping of the surface sediment collected in the collection portion 12, it is desirable to provide the side surface portion 15.

[0015] The attachment portion 17 is a structure for attaching the biological origin nucleic acid collection attachment 10 inside the wheel house 22 of the vehicle 20. In the present embodiment, the attachment portion 17 is a hole structure formed in the wall surface portion 13, and is configured as an attachment hole for attaching the biological origin nucleic acid collection attachment 10 using an attachment member to the inside of the wheel house 22. As the attachment member for attaching the biological origin nucleic acid collection attachment 10 to the inside of the wheel house 22, a rivet, a bolt, a clip, a screw, or the like can be used. In the present embodiment, the attachment portion 17 is provided at each of the four corners of the wall surface portion 13, but the position and number of the attachment portions 17 can be arbitrarily set as long as the biological origin nucleic acid collection attachment 10 can be attached to the inside of the wheel house 22.

[0016] Figure 4 is an explanatory diagram showing how the bio-derived nucleic acid recovery attachment 10 is installed inside the wheelhouse 22. In Figure 4, the installation direction of the bio-derived nucleic acid recovery attachment 10 is indicated by a white arrow. Generally, the wheelhouse 22 is formed from metal members that make up the vehicle body, and a resin fender liner 25 is installed inside the wheelhouse 22 so as to cover the inner wall surface of the wheelhouse 22. The fender liner 25 is attached to the inner wall surface of the wheelhouse 22 using mounting members 26 such as rivets, bolts, clips, and screws. The mounting portion 17 (mounting hole) of the bio-derived nucleic acid recovery attachment 10 in this embodiment is provided so as to be fitted into a position corresponding to the arrangement position of the mounting members 26 for attaching the fender liner 25 to the wheelhouse 22. Therefore, when attaching the fender liner 25 to the wheelhouse 22, the mounting holes 27 of the fender liner 25 and the mounting holes (mounting parts 17) of the bio-derived nucleic acid recovery attachment 10 are aligned, and a common mounting member 26 is used, allowing the fender liner 25 and the bio-derived nucleic acid recovery attachment 10 to be attached to the wheelhouse 22 simultaneously. In this way, by using a common mounting member 26 for attaching the fender liner 25 and the bio-derived nucleic acid recovery attachment 10, the increase in the number of parts required for attaching the bio-derived nucleic acid recovery attachment 10 can be suppressed. However, the mounting member for attaching the bio-derived nucleic acid recovery attachment 10 to the inside of the wheelhouse 22 and the mounting member for attaching the fender liner 25 to the inside of the wheelhouse 22 may be prepared separately. Furthermore, when the bio-derived nucleic acid recovery attachment 10 is attached to the inner wall surface of the wheelhouse 22 to recover surface sediments, the fender liner 25 may be omitted in at least the portion that overlaps with the mounting location of the bio-derived nucleic acid recovery attachment 10 in order to secure mounting space for the bio-derived nucleic acid recovery attachment 10.

[0017] The bio-derived nucleic acid recovery attachment 10 of this embodiment further includes a mesh portion 19 positioned to cover the opening 18 described above. Of the surface sediment kicked up by the tires 24 while the vehicle 20 is in motion, surface sediment larger than the pore diameter of the mesh portion 19 is repelled by the mesh portion 19, and only surface sediment smaller than the pore diameter of the mesh portion 19 can enter the holding space 16. Among the surface sediment, relatively large surface sediment such as pebbles and tree branches are often inefficient and unsuitable as targets for bio-derived nucleic acid extraction. Therefore, the pore diameter of the mesh portion 19 should be appropriately set according to the size of the surface sediment that should be excluded from the target of bio-derived nucleic acid extraction. From this viewpoint, it is desirable that the pore diameter of the mesh portion 19 be, for example, 1 cm or less. With this configuration, it becomes possible to efficiently recover surface sediment that contains a relatively large amount of bio-derived nucleic acid. Here, the pore diameter of the mesh portion 19 refers to the average value of the diameter of the circles inscribed in the pores of the mesh that constitute the mesh portion 19. In addition, the mesh portion 19 is not essential for the biological nucleic acid recovery attachment 10, and it is also possible to omit the mesh portion 19.

[0018] When performing the operation to recover biologically derived nucleic acids, if the biologically derived nucleic acid recovery attachment 10 is to be reused and reused, the biologically derived nucleic acid recovery attachment 10 should be made of a material that allows for repeated processing to suppress the residue of biologically derived nucleic acids from surface sediments that were attached during the previous use. Specifically, processing to suppress the residue of biologically derived nucleic acids attached during the previous use includes washing the biologically derived nucleic acid recovery attachment 10 and nucleic acid removal processing using chlorine-based bleach (e.g., sodium hypochlorite solution) or nucleic acid decomposing agents. From the viewpoint of facilitating such processing to suppress the residue of biologically derived nucleic acids, it is desirable that the biologically derived nucleic acid recovery attachment 10 be made of a polymer material such as stainless steel, polyethylene, silicone, or thermoplastic elastomer (TPE). Among the above materials, it is particularly desirable to construct the biologically derived nucleic acid recovery attachment 10 from a material selected from polyethylene, silicone, and thermoplastic elastomer (TPE). These materials are relatively soft materials among materials that can be subjected to the above processing to suppress the residue of biologically derived nucleic acids. By using a flexible material to construct at least the portion including the wall surface 13, it becomes easier to attach the bio-derived nucleic acid recovery attachment 10 while bending it to conform to the shape of the inner wall surface of the wheelhouse 22. However, the bio-derived nucleic acid recovery attachment 10 may be for single use only, rather than being reused repeatedly.

[0019] When recovering surface sediments using the bio-derived nucleic acid recovery attachment 10, the bio-derived nucleic acid recovery attachment 10 is attached to the inner wall surface of the wheel well 22 of the vehicle 20, as explained with reference to Figure 4. Then, by driving the vehicle 20 through the recovery target area, which is predetermined as an area to be surveyed and monitored for the species of organisms inhabiting the area, the surface sediments present on the ground surface of the recovery target area can be recovered using the bio-derived nucleic acid recovery attachment 10. The vehicle 20 can be of any type, but from the viewpoint of securing space to attach the bio-derived nucleic acid recovery attachment 10 between the tire 24 and the wheel well 22, it is desirable to use a large vehicle such as a truck.

[0020] (A-2) Analysis of biologically derived nucleic acids: By extracting and analyzing bio-derived nucleic acids from surface sediments recovered as described above, it becomes possible to perform analyses such as biological monitoring using bio-derived nucleic acids. An example of a method for extracting nucleic acids from surface sediments is shown below. When extracting nucleic acids, first, at least a portion of the surface sediment held in the holding space 16 of the bio-derived nucleic acid recovery attachment 10 is suspended in a liquid. The liquid used here may be a lysation buffer (Lysis buffer) used to dissolve cells and tissues. The suspension in the liquid may be performed by adding, for example, ceramic beads to the liquid and stirring. This suspends the nucleic acid-containing sediment contained in the surface sediment in the liquid, and the bio-derived nucleic acids in the nucleic acid-containing sediment are eluted into the suspension. Subsequently, the bio-derived nucleic acids are extracted from the suspension from which the bio-derived nucleic acids have been eluted. For example, the extraction of bio-derived nucleic acids can be performed by centrifuging the suspension, recovering the liquid in which the bio-derived nucleic acids have been dissolved as the supernatant, selecting an appropriate column to remove impurities, and then purifying the bio-derived nucleic acids using a column that adsorbs nucleic acids. Examples of the impurities mentioned above include substances that inhibit reactions involved in the PCR process, which will be discussed later.

[0021] Such nucleic acid extraction processes are known methods, and for example, commercially available soil DNA extraction kits may be used. Specifically, when the amount of surface sediment to be extracted is less than approximately 500 mg, NucleoSpin Soil (manufactured by Takara Bio Inc., NucleoSpin is a registered trademark) can be used as the soil DNA extraction kit. Furthermore, when the amount of surface sediment to be extracted is greater than the above and less than approximately 10 g, DNeasy PowerMax Soil Kit (manufactured by Qiagen) can be used as the soil DNA extraction kit.

[0022] The biological nucleic acids extracted in this manner can be analyzed using various known methods. For example, species-specific analyses can be performed, such as determining the presence or absence of a specific biological species or quantifying its abundance using quantitative PCR (qPCR). In this case, by focusing on a specific group of biological species, such as arthropods, microorganisms, birds, or mammals, and selecting appropriate primers to perform PCR, it becomes possible to perform analyses focused on that specific group of biological species. Furthermore, by performing PCR using appropriate universal primers, it is possible to identify a wider range of biological species at once. Alternatively, comprehensive analyses can be performed to identify biological species by comparing the base sequences obtained using a next-generation sequencer with a database, or haplotype analysis can be performed within the same species. In this way, information on organisms inhabiting or residing in the collection area can be obtained.

[0023] As described above, this embodiment uses a bio-derived nucleic acid recovery attachment 10 to recover bio-derived nucleic acids from the ground surface of the recovery area. Therefore, by driving the vehicle 20 in the designated recovery area, ground sediment containing bio-derived nucleic acids can be efficiently recovered from the recovery area. By extracting bio-derived nucleic acids from the recovered ground sediment, comprehensive analyses such as the identification of biological species present in the recovery area can be efficiently performed. In particular, this embodiment recovers ground material kicked up by the tires 24 as the vehicle 20 drives, thus reducing the bias in the locations where samples are collected. For example, when soil is the sample target, the sample collection locations are generally dispersed within the recovery area. However, when collecting ground sediment kicked up by the tires 24 while driving the vehicle 20, as in this embodiment, sampling becomes possible from a wider area that spreads continuously within the recovery area. Therefore, it becomes possible to comprehensively analyze the investigation and monitoring of biological species inhabiting the recovery area.

[0024] Furthermore, in this embodiment, the surface sediments recovered from the target area may include not only nucleic acids derived from organisms living on the surface of the target area, but also nucleic acids of biological origin released into the atmosphere. This is because substances containing nucleic acids of biological origin and having a certain weight, such as biological tissues and excrement, tend to accumulate on the surface. Therefore, according to this embodiment, it becomes possible to analyze various biological species that exist above the surface of the target area. For example, if sampling is done from soil, the obtained nucleic acids of biological origin are likely to be limited to those derived from organisms living in the sampled soil, but according to this embodiment, it becomes possible to recover nucleic acids of biological origin from a wider range of biological species that inhabit or reside in the target area. In particular, in this embodiment, nucleic acid-containing sediments are sampled from a dry environment on land, and since the sampling is done from an environment in which nucleic acids can exist more stably compared to, for example, sampling from a body of water, the recovery efficiency of nucleic acids of biological origin can be increased.

[0025] Furthermore, according to this embodiment, a bio-derived nucleic acid recovery attachment 10 is attached to the inside of the wheel well 22 of the tire 24, and ground sediment is accumulated and recovered in the holding space 16 inside the bio-derived nucleic acid recovery attachment 10. Another method for recovering ground sediment from the area traveled by the vehicle 20 is to recover ground sediment attached to the surface of the tire 24 from the tire surface. According to this embodiment, since a holding space 16 for accumulating ground sediment is formed inside the bio-derived nucleic acid recovery attachment 10, it is possible to recover more ground sediment while suppressing the shedding of ground sediment once it has been recovered, compared to, for example, recovering ground sediment attached to the tire surface as described above. As a result, it becomes possible to set a wider area for recovering ground sediment and to set a longer travel distance for the vehicle 20 to recover ground sediment.

[0026] Furthermore, according to this embodiment, since the bio-derived nucleic acid recovery attachment 10 is used to recover surface sediment kicked up by the tire 24, unlike the case of recovering surface sediment attached to the tire surface as described above, it becomes possible to recover a wider variety of surface sediment, including large grains of sand that do not easily adhere to the tire. Also, when recovering surface sediment attached to the tire surface as described above, the ease with which surface sediment adheres may differ depending on the material constituting the tire surface, but when recovering surface sediment kicked up by the tire 24 as in this embodiment, the influence of the material constituting the tire surface can be suppressed. Also, when recovering surface sediment attached to the tire surface as described above, the amount of surface sediment that can be recovered changes depending on the size of the tire, i.e., the surface area of ​​the tire, but according to this embodiment, the influence of the size of the tire on the amount of surface sediment that can be recovered can be suppressed. Furthermore, when recovering surface sediment attached to the tire surface as described above, it is affected by the condition of the ground due to weather, etc., and for example, the recovery efficiency of surface sediment may decrease in bad weather, but according to this embodiment, the influence of the condition of the ground due to weather, etc. on the recovery efficiency of surface sediment can be suppressed. Therefore, when using a vehicle to recover surface deposits, it is desirable to use either the method using the bio-derived nucleic acid recovery attachment 10 of this embodiment or the method of recovering surface deposits attached to the tire surface as described above, depending on conditions such as the size of the area to be recovered, the vehicle's travel time, the condition of the ground surface, the type and size of the vehicle, and the ease of the recovery operation. This makes it possible to recover surface deposits and bio-derived nucleic acids more appropriately.

[0027] In the following section, to demonstrate the usefulness of recovering and analyzing biologically derived nucleic acids using the biologically derived nucleic acid recovery attachment 10 of this embodiment, we present, as a reference example, the results of recovering ground sediment attached to the inside of the wheelhouse 22 (surface of the fender liner 25), extracting biologically derived nucleic acids, and identifying the species. Here, as another reference example, we also present the results of recovering ground sediment attached to the tire surface of the vehicle 20, similarly extracting biologically derived nucleic acids, and identifying the species.

[0028] The recovery and analysis of biological nucleic acids were performed in February 2022 using a regular vehicle. The weather on the day of sampling was light rain. After driving the vehicle 20 through the designated area for recovery, soil sediment adhering to the inside of the wheel wells 22 and the tire surface was wiped with a swab, and DNA was extracted from the swabs. For DNA extraction, a commercially available soil sample DNA extraction kit (NucleoSpin Soil, Takara Bio Inc.) (NucleoSpin is a registered trademark) was used as the extraction kit. The nucleic acid extracts obtained using the above kit were used as DNA samples from the inside of the wheel wells and from the tire surface, respectively, and the 12S rRNA sequence was amplified by PCR using a predetermined primer (existing universal primers for mammals and birds). The Kapa Hifi PCR Kit (KAPABIOSYSTEMS) was used for amplification. Furthermore, PCR was performed to add an index for sequence analysis, and the PCR product was purified by beads. Subsequently, the sequence was obtained using a next-generation sequencer. The sequencer used was the Illumina iSeq100 (manufactured by Illumina Corporation) (iSeq is a registered trademark).

[0029] Figure 5 is an explanatory diagram showing the results of the analysis of the DNA sample from the inside of the wheel well, and Figure 6 is an explanatory diagram showing the results of the analysis of the DNA sample from the tire surface. For the obtained sequences, species were extracted using the MiFish Pipeline, a DNA analysis pipeline, and further species identification was attempted by performing a BLAST homology search on the obtained sequences. For this analysis, three samples were taken from the inside of the wheel well and three from the tire surface of the same vehicle on the same day, and species detected in one or more locations were listed. Figures 5 and 6 show the results of species identification at the genus level.

[0030] As shown in Figure 5, many species of both birds and mammals were detected in the DNA sample from the inside of the wheelhouse. Thus, since many species of organisms were detected from the surface sediment attached to the inner wall surface of the wheelhouse 22, it is thought that using the biological nucleic acid recovery attachment 10 of this embodiment will make it possible to efficiently recover a larger amount of surface sediment.

[0031] In contrast, the number of species detected from DNA samples on the tire surface was small for both birds and mammals. The small number of species detected from the DNA samples on the tire surface is thought to be due to the influence of the wet ground surface (road surface) caused by light rain on the day of sampling, and the detachment of ground deposits that had initially adhered due to friction with the road surface. Thus, it was confirmed that by collecting ground deposits splashed up from the tires into the inside of the wheel well, it is possible to efficiently recover biological nucleic acids while minimizing the influence of the ground surface conditions, and to perform analysis of biological nucleic acids.

[0032] B. Second Embodiment: Figure 7 is an explanatory diagram showing the configuration of the biological nucleic acid recovery attachment 110 of the second embodiment, in the same manner as in Figure 3. The biological nucleic acid recovery attachment 110 is used in the same manner as the biological nucleic acid recovery attachment 10 of the first embodiment. In the second embodiment, the same reference numerals are used for parts common to the first embodiment, and the differences from the biological nucleic acid recovery attachment 10 of the first embodiment will be described below.

[0033] The biological nucleic acid recovery attachment 110 of the second embodiment does not have a mesh portion 19 and instead of the collection portion 12 of the first embodiment, it is equipped with a collection portion 112. The collection portion 112 includes a wall portion 13, a retention space forming portion 14, a side portion 15, and further comprises a porous body 130 arranged to fill the retention space 16. The porous body 130 can be, for example, a relatively coarse sponge-like porous body with a pore diameter of about 2 mm to 1 cm. There are no particular restrictions on the material constituting the porous body 130 or its hardness, as long as it can hold the ground sediment kicked up by the tire within the pores inside the porous body 130. However, in order to suppress nonspecific amplification when performing PCR during the analysis of nucleic acids extracted from the recovered ground sediment, it is desirable that the porous body 130 be formed from a non-biological material such as synthetic resin rather than a biological material. Even when using the bio-derived nucleic acid recovery attachment 110 of this second embodiment, the same effects as in the first embodiment can be obtained by holding the ground sediment kicked up by the tires 24 of the vehicle 20 in the pores inside the porous body 130.

[0034] C. Third Embodiment: Figure 8 is an explanatory diagram showing the configuration of the biological nucleic acid recovery attachment 210 of the third embodiment, in the same manner as in Figure 3. The biological nucleic acid recovery attachment 210 is used in the same manner as the biological nucleic acid recovery attachment 10 of the first embodiment. The biological nucleic acid recovery attachment 210 of the third embodiment comprises a porous body 230 and a frame portion 232.

[0035] The porous body 230 can be formed with the same pore size as the porous body 130 of the second embodiment using the same material. The porous body 230 only needs to be shaped to be attached to the inside of the wheelhouse 22 so as to follow the inner wall surface of the wheelhouse 22, but Figure 8 shows the porous body 230 as a plate shape with each face being rectangular.

[0036] The frame portion 232 is a frame-shaped member with a space formed inside for housing the porous body 230. It is composed of a framework provided along each side of the plate-shaped porous body 230, and has a structure in which the parts corresponding to each surface of the plate-shaped porous body 230 are open. The porous body 230 is held in place by fitting it into the inside of such a frame portion 232. When the frame portion 232 is reused repeatedly when performing the operation of recovering biological nucleic acids, the frame portion 232 may be made of the same material as the constituent material of the biological nucleic acid recovery attachment 10 described in the first embodiment, so as a material that can be repeatedly treated to suppress the residue of biological nucleic acids contained in the surface sediment attached during the previous use. However, the frame portion 232 may be for single use only.

[0037] The frame portion 232 includes mounting portions (not shown) for attaching the frame portion 232 to the inside of the wheelhouse 22 of the vehicle 20. The mounting portions can be provided, for example, in the part of the framework portion constituting the frame portion 232 that is in contact with the inside of the wheelhouse 22, and can be configured as mounting holes for attaching the frame portion 232 to the inside of the wheelhouse 22 using mounting members. The mounting members used can be the same as those used for mounting member 26 in the first embodiment. The position of the mounting portions can be set to correspond to the position of mounting members for attaching the fender liner to the wheelhouse, thereby reducing the number of parts involved in the attachment.

[0038] When using the bio-derived nucleic acid recovery attachment 210 of this third embodiment, the same effects as the first and second embodiments can be obtained by holding the ground deposits kicked up by the tires 24 of the vehicle 20 in the pores inside the porous body 230. Furthermore, according to the bio-derived nucleic acid recovery attachment 210 of the third embodiment, a frame portion 232 is used to support the porous body 230 without having a wall portion 13, a holding space forming portion 14, and a side portion 15. Therefore, compared to the bio-derived nucleic acid recovery attachment 110 of the second embodiment, the entire bio-derived nucleic acid recovery attachment can be made thinner and lighter, making it easier to position the bio-derived nucleic acid recovery attachment between the inner wall surface of the wheelhouse 22 and the tire 24.

[0039] Unlike the third embodiment shown in Figure 8, it is also possible to have a configuration that does not use the frame portion 232. That is, instead of supporting the porous body 230 with special members such as the frame portion 232, the porous body 230 may be directly attached to the inside of the wheelhouse 22 by, for example, providing mounting holes in the porous body 230 and using the mounting members 26 described above.

[0040] When recovering surface sediments using a porous material, as in the second or third embodiment, in order to extract biological nucleic acids from the surface sediments, first, the porous material holding the surface sediments is washed in a liquid, and the held surface sediments are transferred to the liquid to obtain a suspension. Here, the liquid used to suspend the surface sediments should be a nuclease-free buffer or nuclease-free water that does not contain nucleic acid-degrading enzymes. Biological nucleic acids can be extracted from the suspension obtained in this way. The extraction of biological nucleic acids can be carried out by known methods involving steps such as elution of nucleic acids from the surface sediments in the suspension, recovery of the solution from which the nucleic acids have been eluted, and removal of impurities using an appropriate column. Such extraction of biological nucleic acids can be easily carried out using a commercially available DNA extraction kit for environmental water samples. Specifically, for example, NucleoSpin eDNA Water (manufactured by Takara Bio Inc., NucleoSpin is a registered trademark) can be used.

[0041] Furthermore, prior to extracting biological nucleic acids from surface sediments held in a porous material, the surface sediments attached to the porous material may be concentrated. In this case, first, the porous material holding the surface sediments is washed in a liquid such as a nuclease-free buffer or nuclease-free water to obtain a suspension of surface sediments. Then, the obtained suspension is filtered to obtain a residue containing surface sediments, thereby concentrating the surface sediments on the filter. The pore size of the filter used should be appropriately selected to efficiently recover the surface sediments, for example, 0.22 to 10 μm. During filtration, the filtration efficiency can be increased by, for example, creating negative pressure using a vacuum pump or increasing pressure using a pump.

[0042] After obtaining a filter containing concentrated surface sediments, this filter is crushed and suspended in a lysing buffer (Lysis buffer) used to dissolve cells and tissues, thereby eluting the biological nucleic acids from the surface sediments into the buffer. The biological nucleic acids can then be extracted using known methods, which include steps such as recovering the solution containing the eluted nucleic acids and removing impurities using an appropriate column. Extraction of biological nucleic acids from such filters can be easily performed using commercially available DNA extraction kits for soil samples; specifically, for example, NucleoSpin Soil (manufactured by Takara Bio Inc., NucleoSpin is a registered trademark) can be used.

[0043] After extracting biological nucleic acids from surface sediments held by the porous material as described above, the extracted biological nucleic acids can be analyzed to, for example, identify the species of organism, as explained in the first embodiment.

[0044] D. Other embodiments: In the embodiments described above, the bio-derived nucleic acid recovery attachment for recovering bio-derived nucleic acids is attached to the rear side of the wheelhouse 22, but it may be attached to a different location inside the wheelhouse 22. For example, it can be attached to the upper or front side of the wheelhouse 22. However, the configuration in which the bio-derived nucleic acid recovery attachment is attached to the rear side of the wheelhouse 22, as in the embodiments, is desirable because it allows for the direct recovery of ground sediment kicked up by the tire. If the bio-derived nucleic acid recovery attachment is installed in another location inside the wheelhouse 22, it is more likely that ground sediment that has adhered to the tire and then detached from the tire will be recovered. Thus, the amount of ground sediment that detaches from the tire towards a specific location inside the wheelhouse 22 after adhering to the tire is less than the amount of ground sediment that is kicked up towards the rear side of the vehicle by the tire 24. Furthermore, as previously described, the efficiency of ground sediment adhesion to the tire is more strongly affected by factors such as weather and ground conditions. As a result, if the bio-derived nucleic acid recovery attachment is placed anywhere other than the rear of the vehicle, the recovery efficiency of surface sediments by the bio-derived nucleic acid recovery attachment will also be affected by the above-mentioned factors. By attaching the bio-derived nucleic acid recovery attachment to the rear of the inner wall surface of the wheelhouse 22, as in the embodiment, and directly recovering the surface sediments that have been thrown up, it becomes possible to recover more surface sediments efficiently and stably while suppressing the above-mentioned effects.

[0045] In the embodiments described above, the mounting portion provided on the biological nucleic acid recovery attachment was a mounting hole for attaching the biological nucleic acid recovery attachment to the inside of the wheelhouse 22 using mounting members such as rivets, bolts, clips, and screws, but a different configuration is also possible. The mounting portion only needs to be a structure that allows the biological nucleic acid recovery attachment to be attached to the inside of the wheelhouse 22. For example, without using a separate mounting member, the biological nucleic acid recovery attachment may be directly fitted into the fender liner 25 inside the wheelhouse 22, and a structure that can engage with each other may be provided on both the biological nucleic acid recovery attachment and the fender liner 25. In this case, the above-mentioned engageable structure provided on the biological nucleic acid recovery attachment side becomes the mounting portion.

[0046] This disclosure is not limited to the embodiments described above, and can be implemented in various configurations without departing from its spirit. For example, the technical features in the embodiments corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate 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 deleted as appropriate.

[0047] This disclosure can also be implemented in the following forms: [Application Example 1] A bio-derived nucleic acid recovery attachment, The aforementioned biological nucleic acid recovery attachment includes a mounting portion for attaching it to the inside of the wheel well of a tire on a vehicle, A collection unit that collects and holds biologically derived nucleic acids that are kicked up by the tires as the vehicle moves, A bio-based nucleic acid recovery attachment equipped with [specific feature]. [Application Example 2] The biological nucleic acid recovery attachment described in Application Example 1, The aforementioned collection unit is A wall portion attached to the wheel house along the inner wall surface of the wheel house, When the bio-derived nucleic acid recovery attachment is mounted inside the wheel well, a holding space forming part is located below the wall surface and forms a holding space between itself and the wall surface for holding the bio-derived nucleic acid, An opening is provided at a position opposite to the tire, which connects the holding space with the outside of the biological nucleic acid recovery attachment, A bio-based nucleic acid recovery attachment equipped with [specific feature]. [Application Example 3] The biological nucleic acid recovery attachment described in Application Example 2, further, The opening is covered by a mesh portion. Bio-derived nucleic acid recovery attachment. [Application Example 4] The biological nucleic acid recovery attachment described in Application Example 3, The diameter of the holes in the mesh portion is 1 cm or less. Bio-derived nucleic acid recovery attachment. [Application Example 5] A biological nucleic acid recovery attachment described in any one of the application examples 1 to 4, The collection unit comprises a porous body having pores formed therein that allow the biological nucleic acids to be taken in. Bio-derived nucleic acid recovery attachment. [Application Example 6] The bio-derived nucleic acid recovery attachment described in Application Example 5, further, The frame comprises a space for housing the porous material and a frame portion on which the mounting portion is provided. Bio-derived nucleic acid recovery attachment. [Application Example 7] A biological nucleic acid recovery attachment described in any one of the application examples 1 to 6, The mounting portion includes mounting holes provided at positions corresponding to the placement positions of mounting members for attaching the fender liner to the wheel well, into which the mounting members are fitted. Bio-derived nucleic acid recovery attachment. [Explanation of Symbols]

[0048] 10,110,210… Bio-derived nucleic acid recovery attachments 12,112... Collection section 13…Wall section 14...Holding space forming part 15...Side part 16...Holding space 17…Mounting part 18…Opening 19... Mesh part 20... Vehicles 22... Wheelhouse 24... Tires 25…Fender Liner 26…Mounting parts 27…Mounting holes 130,230…Porous material 232...Frame body part

Claims

1. A bio-derived nucleic acid recovery attachment, The aforementioned biological nucleic acid recovery attachment includes a mounting portion for attaching it to the inside of the wheel well of a tire on a vehicle, A collection unit that collects and holds biologically derived nucleic acids that are kicked up by the tires as the vehicle moves, Equipped with, The aforementioned collection unit is A wall portion attached to the wheel house along the inner wall surface of the wheel house, When the bio-derived nucleic acid recovery attachment is mounted inside the wheel well, a holding space forming part is located below the wall surface and forms a holding space between itself and the wall surface for holding the bio-derived nucleic acid, An opening is provided at a position opposite to the tire, which connects the holding space with the outside of the biological nucleic acid recovery attachment, A bio-based nucleic acid recovery attachment equipped with [specific feature].

2. A biological nucleic acid recovery attachment according to claim 1, further, The opening is covered by a mesh portion. Bio-derived nucleic acid recovery attachment.

3. A biological nucleic acid recovery attachment according to claim 2, The diameter of the holes in the mesh portion is 1 cm or less. Bio-derived nucleic acid recovery attachment.

4. A biological nucleic acid recovery attachment according to claim 1, The collection unit comprises a porous body having pores formed therein that allow the biological nucleic acids to be taken in. Bio-derived nucleic acid recovery attachment.

5. A bio-derived nucleic acid recovery attachment, The aforementioned biological nucleic acid recovery attachment includes a mounting portion for attaching it to the inside of the wheel well of a tire on a vehicle, A collection unit that collects and holds biologically derived nucleic acids that are kicked up by the tires as the vehicle moves, Equipped with, The mounting portion includes mounting holes provided at positions corresponding to the placement positions of mounting members for attaching the fender liner to the wheel well, into which the mounting members are fitted. Bio-derived nucleic acid recovery attachment.

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