Odor detection kit, method for manufacturing odor detection kit, and odor detection method

Abstract

An odor detection kit for a fluorometer, said odor detection kit comprising: a substrate 10; and odor detection cells 20 that are positioned in a single layer on the substrate 10, have olfactory receptors, and express a fluorescent protein, the fluorescence intensity thereof changing in accordance with the concentration of odor molecules.

Description

[Technical field]

[0001] The present invention relates to detection technology, and to a method for manufacturing an odor detection kit and a method for detecting an odor. [Background technology]

[0002] Water is obtained from a water source, treated at a water purification plant, and then supplied as tap water to homes, stores, and businesses. Alternatively, food factories and the like obtain water from their own water sources and use the water to produce beverages and food. Examples of water sources include dam lakes, natural lakes and marshes, rivers, and groundwater. When the temperature of the water source rises, planktonic algae and actinomycetes may grow, causing the water obtained from the water source to have a moldy odor. The moldy odor of water may not disappear even after purification. Therefore, it is desirable to quantify the moldy odor of water before supplying it to consumers.

[0003] The level of moldy odor in water can be accurately quantified by gas chromatography mass spectrometry (GC / MS). However, because GC / MS takes time to perform, when water purification and supply are performed continuously, even if GC / MS detects a moldy odor above the specified level, the water from which the sample was taken may already have been supplied to the consumer. In addition, because it is difficult to move the equipment that performs GC / MS, the equipment that performs GC / MS cannot be used to evaluate the moldy odor of water immediately after it is obtained from an outdoor water source on-site. Furthermore, although inspectors can perform a sensory evaluation of the moldy odor of water, it takes time to train the inspectors, and the results of the sensory evaluation may vary depending on the inspector.

[0004] Therefore, simple odor testing methods different from GC / MS have been proposed (see, for example, Patent Documents 1 to 3). However, these methods may not be able to stably and accurately test odors. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 5127783 [Patent Document 2] Patent No. 6474945 [Patent Document 3] Special Publication No. 2002-518035 Summary of the Invention [Problem to be solved by the invention]

[0006] There is a demand for a technique that can easily and stably detect not only the mold odor of water but also the odors of various test subjects. Therefore, an object of the present invention is to provide an odor detection kit, a manufacturing method for an odor detection kit, and an odor detection method that can easily and stably detect odors. [Means for solving the problem]

[0007] According to an aspect of the present invention, there is provided an odor detection kit for a fluorometer comprising a substrate and odor detection cells arranged in a monolayer on the substrate, the odor detection cells having olfactory receptors and expressing a fluorescent protein whose fluorescence intensity changes depending on the concentration of odor molecules.

[0008] In the odor detection kit, the odor detecting cells may be immobilized on the substrate via a cell membrane modifying agent.

[0009] The odor detection kit may further include a tube capable of fixing the substrate therein.

[0010] In the odor detection kit, the odor detection cells may express an olfactory receptor via an introduced gene.

[0011] In the odor detection kit, the odor detection cells may express an insect olfactory receptor.

[0012] In the odor detection kit, the olfactory receptor may be an ionotropic receptor.

[0013] In the odor detection kit, the fluorescent protein may change its fluorescence intensity depending on the ion concentration.

[0014] In the above odor detection kit, the olfactory receptor may be selected from BmOR1, BmOR3, DmOr13a, DmOr56a, DmOr82a, DmOr49b, DmOr85b, and PxOR1.

[0015] In the odor detection kit, the odor detection cells may be insect cells.

[0016] In the odor detection kit, the insect cells may be cells derived from a moth.

[0017] In the odor detection kit, the insect cells may be selected from Sf21, Sf9, High Five, and Tni-derived cells.

[0018] In the odor detection kit, the insect cells may be cells derived from Drosophila melanogaster.

[0019] In the odor detection kit, the insect cells may be Drosophila S2 cells.

[0020] In the odor detection kit, the substrate may be transparent.

[0021] According to another aspect of the present invention, there is provided a method for manufacturing an odor detection kit for a fluorometer, the method including: preparing odor detection cells that have olfactory receptors and express a fluorescent protein whose fluorescence intensity changes depending on the concentration of odor molecules; preparing a substrate; and arranging the odor detection cells in a single layer on the substrate.

[0022] In the above-mentioned method for producing an odor detection kit, the odor detecting cells may be immobilized on the substrate via a cell membrane modifying agent.

[0023] The method for producing the odor detection kit may further include fixing the substrate on which the odor detection cells are arranged in a tube.

[0024] The manufacturing method for the above-mentioned odor detection kit may further include performing steps (a) to (c) multiple times: (a) selecting a portion of cells from a group of cells that have olfactory receptors and express a fluorescent protein, (b) proliferating the selected cells, and (c) confirming the responsiveness of the proliferated cells to an odorant, and selecting proliferated cells whose responsiveness to the odorant is equal to or greater than a standard value as odor detection cells.

[0025] The manufacturing method for the above-mentioned odor detection kit may further include performing steps (a) to (c) multiple times: (a) selecting a portion of cells from a group of cells that have olfactory receptors and express a fluorescent protein, (b) proliferating the selected cells, and (c) confirming the responsiveness of the proliferated cells to an odorant, and selecting the proliferated cells that are most responsive to the odorant as odor detection cells.

[0026] The method for producing the odor detection kit may include expressing a second olfactory receptor in a cell expressing a first olfactory receptor. The first olfactory receptor and the second olfactory receptor may be different. The first olfactory receptor and the second olfactory receptor may react to different odor molecules.

[0027] In the above-mentioned method for producing an odor detection kit, the selected portion of cells may be a single cell.

[0028] According to another aspect of the present invention, there is provided an odor detection method comprising: contacting a fluid to be subjected to odor testing with odor detection cells arranged in a single layer on a substrate, the odor detection cells having olfactory receptors and expressing a fluorescent protein whose fluorescence intensity changes depending on the concentration of odor molecules, and measuring the fluorescence emitted by the odor detection cells.

[0029] In the odor detection method described above, the odor detecting cells may be immobilized on the substrate via a cell membrane modifying agent.

[0030] In the odor detection method, the substrate may be fixed within a tube, and the odor detection method may further include introducing a fluid into the tube.

[0031] In the odor detection method described above, the fluid may be a liquid. Effect of the Invention

[0032] According to the present invention, it is possible to provide an odor detection kit capable of simply and stably detecting an odor, a method for manufacturing an odor detection kit, and an odor detection method. [Brief description of the drawings]

[0033] [Figure 1] FIG. 1 is a schematic diagram of an odor detection kit for a fluorometer according to an embodiment. [Diagram 2] FIG. 2 is a schematic diagram showing a method for expressing an olfactory receptor in an odor detection cell according to an embodiment. [Diagram 3] 1A to 1C are schematic diagrams showing a method for immobilizing odor detecting cells on a substrate according to an embodiment. [Figure 4] 1A and 1B are schematic diagrams showing a method for homogenizing odor detecting cells according to an embodiment. [Diagram 5] FIG. 2 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. [Figure 6] FIG. 2 is a schematic diagram showing a method for expressing multiple olfactory receptors in odor detection cells according to an embodiment. [Figure 7] FIG. 2 is a schematic diagram showing the response of an odor detection cell to an odor molecule according to an embodiment. [Figure 8] 1 shows photographs and graphs illustrating the concentration-dependent response of odor detection cells to odor molecules according to an embodiment. [Figure 9] 1 is a graph showing the specific response of odor detection cells to odor molecules according to an embodiment. [Figure 10] 1 is a graph showing the change over time in fluorescence emitted by a suspension of odor detection cells in an embodiment. [Figure 11] 1 is a graph showing the response of homogenized and non-homogenized odor detection cells to odor molecules according to an embodiment. [Figure 12] 1 is a graph showing the response of odor detection cells subcultured over a long period of time in an embodiment to odor molecules. [Figure 13] 13 is a graph showing the response of odor detection cells that have been subcultured for a long period of time after cryopreservation for three and a half months in an embodiment to odor molecules. [Figure 14] 13 is a graph showing the response of odor detection cells that have been subcultured for a long period of time after cryopreservation for six and a half months in an embodiment to odor molecules. [Figure 15] 1 is a graph showing the measurement results using an odor detection kit according to an embodiment. [Figure 16] 1 is a graph showing the relationship between geosmin concentration and the rate of change in detected fluorescence intensity in an example. [Figure 17] 1 is a graph showing the odor molecule specificity of an odor detection kit according to an embodiment. [Figure 18] 1 is a graph showing a comparison of the time taken for a cell suspension to become stable with an odor detection kit according to an embodiment. [Figure 19] 1 is a graph showing a comparison of the responsiveness to odor molecules of an odor detection kit according to an embodiment and a cell suspension. [Figure 20] 1 is a graph showing a comparison of the variation in responsiveness to odor molecules between an odor detection kit according to an embodiment and a cell suspension. [Figure 21] 1 is a graph showing the results of outdoor measurements using an odor detection kit according to an embodiment. [Figure 22] 1 is a graph showing the change over time in fluorescence emitted by an odor detection cell in an embodiment. [Diagram 23] 1 is a graph showing the change over time in fluorescence emitted by an odor detection cell in an embodiment. [Figure 24] 1 is a graph showing the change over time in fluorescence emitted by an odor detection cell in an embodiment. [Diagram 25] 1 is a graph showing the relationship between the change in fluorescence emitted by an odor detection cell in an embodiment and the concentration of an odor substance. [Figure 26] 1 is a graph showing the change over time in fluorescence emitted by an odor detection cell in an embodiment. [Figure 27] 1 is a graph showing the relationship between the change in fluorescence emitted by an odor detection cell in an embodiment and the concentration of an odor substance. [Figure 28] 1 is a graph showing the relationship between the change in fluorescence emitted by an odor detection cell in an embodiment and an odor substance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] An embodiment of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions, etc. should be determined in light of the following description. In addition, the drawings naturally include parts with different dimensional relationships and ratios.

[0035] As shown in Figure 1, the odor detection kit for a fluorometer according to the embodiment comprises a substrate 10 and odor detection cells 20 arranged in a single layer on the substrate 10, the odor detection cells 20 having olfactory receptors and expressing fluorescent proteins whose fluorescence intensity changes depending on the concentration of odor molecules.

[0036] The substrate 10 is, for example, transparent and made of glass, resin, or the like.

[0037] The odor detection cell 20 expresses an olfactory receptor on the cell membrane. In the odor detection cell 20, the olfactory receptor may be naturally expressed or may be expressed by an introduced gene. The olfactory receptor may be an insect olfactory receptor.

[0038] The odor detection cell 20 may be an insect cell. The insect cell may be a cell derived from a moth such as Spodoptera frugiperda or Trichoplusia ni. Examples of cells derived from the armyworm moth include Sf21 and Sf9. Sf21 cells are derived from ovarian cells. Sf21 cells divide infinitely, and it is possible to establish a stable expression line that permanently expresses an introduced gene. Sf21 cells can survive in a wide temperature range from 18°C ​​to 40°C, and do not require carbon dioxide to adjust the pH of the culture solution. Sf21 cells do not originally have olfactory receptors, but it is possible to express olfactory receptors by introducing an olfactory receptor gene. Sf9 cells are a clone of Sf21. Examples of cells derived from the armyworm moth include High Five and Tni. Tni-derived cells are derived from ovarian cells.

[0039] Alternatively, the insect cell may be a cell derived from Drosophila, such as a Drosophila S2 cell.

[0040] The olfactory receptor may be a G protein-coupled receptor or an ionotropic receptor. An ionotropic receptor has a site that interacts with a ligand, which is an odor molecule, and a site where ions flow in. When the ionotropic receptor of the odor detection cell 20 binds to a ligand, positive ions such as sodium ions and calcium ions flow into the odor detection cell 20. In the odor detection cell 20, the influx of ions can occur within about several tens of milliseconds after the binding of the ligand. The amount of ions that flow in is large, and the amount of ions that flow into the cell for each binding of a ligand is about 10 7 It is also called "pieces."

[0041] In general, a particular type of olfactory receptor has specificity for a particular odor molecule. In the odor detection cell 20, only one type of olfactory receptor corresponding to one type of odor molecule may be expressed, or multiple types of olfactory receptors corresponding to multiple types of odor molecules may be expressed. In addition, the amount of olfactory receptors to be expressed may be adjusted.

[0042] Examples of olfactory receptors include, but are not limited to, DmOr56a, a Drosophila receptor for geosmin, which has a moldy odor; DmOr82a, a Drosophila receptor for geranyl acetate, which has a fragrant or fruity odor; DmOr49b, a Drosophila receptor for 2-methylphenol (o-cresol), which has the odor of human sweat; DmOr13a, a Drosophila receptor for 1-octen-3-ol, which has a moldy odor; BmOR1, a Bombykol receptor, which is a sex pheromone of the silkworm moth; BmOR3, a Bombykal receptor, which is a subcomponent of the sex pheromone of the silkworm moth; DmOr85b, a general odor receptor of Drosophila melanogaster; and PxOR1, a sex pheromone receptor of the diamondback moth. Note that "geosmin" is also called "geosmin".

[0043] When expressing an olfactory receptor in an odor detection cell 20 by genetic engineering, for example, as shown in FIG. 2, a gene encoding the olfactory receptor is inserted into a vector, and the constructed vector is transfected into a host cell. The gene encoding the olfactory receptor can be isolated, for example, by extracting mRNA from an insect's olfactory organ and synthesizing cDNA. From the isolated cDNA, a part of the gene encoding the olfactory receptor can be amplified by PCR using PCR primers.

[0044] A portion of the gene encoding the olfactory receptor can also be obtained by inserting a synthesized double-stranded cDNA into an appropriate vector and using the vector to transform E. coli or the like to prepare a cDNA library. The cDNA can be inserted into a vector by a conventional method using a restriction enzyme and ligase, for example, by cleaving the obtained cDNA with a restriction enzyme, inserting it into the restriction enzyme site of the vector DNA, and ligating it to the vector.

[0045] A fluorescent protein is expressed in the odor detection cell 20. As described above, when an odor molecule binds to an ionotropic olfactory receptor in the odor detection cell 20, ions flow into the odor detection cell 20. Therefore, by introducing a gene that expresses a fluorescent protein whose fluorescence intensity changes depending on ions into the odor detection cell 20, it is possible to confirm whether the odor detection cell 20 is detecting an odor molecule from the change in fluorescence intensity. Examples of fluorescent proteins include GCaMP3, GCaMP6s, and aequorin, which react with calcium ions to emit fluorescence.

[0046] When the concentration of odor molecules is high, the concentration of ions flowing into the odor detection cells 20 is also high, and many fluorescent proteins emit fluorescence. As a result, the intensity of the fluorescence emitted by the odor detection cells 20 is strong. When the concentration of odor molecules is low, the concentration of ions flowing into the odor detection cells 20 is also low, and fewer fluorescent proteins emit fluorescence. As a result, the intensity of the fluorescence emitted by the odor detection cells 20 is weak. Therefore, it is possible to evaluate the concentration of odor molecules based on the fluorescence intensity.

[0047] In addition, the interaction between odor molecules and odor receptors is weak, and it is believed that the same odor molecule interacts with the same odor receptor multiple times, the same odor molecule interacts with multiple odor receptors in the same cell, or the same odor molecule interacts with the odor receptors in multiple different cells. Therefore, even if the number of odor molecules is small, ion influx into the cell occurs repeatedly, making it possible to detect odor molecules with high sensitivity.

[0048] The surface of the substrate 10 shown in Fig. 1 may be coated with a protein such as collagen or bovine serum albumin (BSA). The odor detecting cells 20 may be immobilized on the substrate 10 via a protein covering the surface of the substrate 10. Alternatively, the odor detecting cells 20 may be immobilized on the substrate 10 via a cell membrane modifying agent. Alternatively, as shown in Fig. 3, the odor detecting cells 20 may be immobilized on the substrate 10 via a cell membrane modifying agent and a protein such as collagen or BSA covering the surface of the substrate 10.

[0049] The cell membrane modifying agent has a cell membrane binding group that binds to the cell membrane of the odor detecting cell 20. A lipid can be used as the cell membrane binding group. For example, an oleyl group, a dioleyl group, a cholesterol group, and a tetraethylene glycol group can be used as the cell membrane binding group. The cell membrane modifying agent also has a substrate binding group that binds to the surface of the substrate 10 or to the protein that covers the substrate 10. An N-hydroxysuccinimide (NHS) group can be used as the substrate binding group that binds to the protein that covers the substrate 10. The cell membrane modifying agent may have polyethylene glycol (PEG) between the cell membrane binding group and the substrate binding group to increase water solubility. In order to provide an appropriate distance between the substrate 10 and the odor detecting cell 20, the number of repeating ethylene oxide units of PEG may be appropriately set.

[0050] An example of the cell membrane modifying agent is oleyl-O-poly(ethylene glycol) succinyl-N-hydroxy-succinimidyl ester (oleyl-PEG-NHS) represented by the following chemical formula (1), where n is a natural number. [ka]

[0051] 1, the odor detection kit for a fluorometer according to the embodiment further includes a tube 30 capable of fixing the substrate 10 therein. For example, by making the inner diameter of the tube 30 and the width of the substrate 10 approximately the same, it is possible to prevent the substrate 10 inserted in the tube 30 from moving within the tube 30.

[0052] When odor detecting cells 20 are non-adhesive cells, even if odor detecting cells 20 are brought into contact with the surface of substrate 10, odor detecting cells 20 may not adhere to the surface of substrate 10. Alternatively, even if odor detecting cells 20 are adhesive cells, they may have a weak adhesive force to substrate 10 and may easily peel off from substrate 10. In contrast, by using a cell membrane modifying agent, odor detecting cells 20 can be fixed in a single layer on the surface of substrate 10 even if odor detecting cells 20 are non-adhesive cells or adhesive cells with weak adhesive force.

[0053] Next, a method for manufacturing an odor detection kit for a fluorometer according to an embodiment will be described. The method for manufacturing an odor detection kit for a fluorometer according to an embodiment includes preparing odor detection cells 20 that have olfactory receptors and express a fluorescent protein whose fluorescence intensity changes depending on the concentration of odor molecules, preparing a substrate 10, and arranging the odor detection cells 20 in a single layer on the substrate 10.

[0054] The odor detection cells 20 may be selected from the proliferated cells whose responsiveness to the odorant is equal to or greater than a reference value by carrying out steps (a) to (c) multiple times: (a) selecting a portion of cells from a group of cells that have an olfactory receptor and express a fluorescent protein, (b) growing the selected cells, and (c) confirming the responsiveness of the grown cells to the odorant. The cells selected in step (a) may be single cells.

[0055] Alternatively, the odor detection cells 20 may be selected from the proliferated cells having the highest responsiveness to the odorant by performing steps (a) to (c) multiple times: (a) selecting a portion of cells from a group of cells having an olfactory receptor and expressing a fluorescent protein, (b) growing the selected cells, and (c) confirming the responsiveness of the grown cells to the odorant. The cell selected in step (a) may be a single cell.

[0056] Specifically, as shown in FIG. 4, a cell line may be established by repeatedly diluting a group of cell lines that have an olfactory receptor and express a fluorescent protein to select a single or a small number of cells, and culturing and expanding the selected cells. By carrying out this process multiple times, multiple cell lines are established. Of the multiple established cell lines, a cell line whose responsiveness to an odorant is equal to or exceeds a predetermined reference value may be used as the odor detecting cell 20. Alternatively, of the multiple established cell lines, a cell line whose responsiveness to an odorant is the highest may be used as the odor detecting cell 20.

[0057] As shown in FIG. 5 and FIG. 6, a cell expressing an established olfactory receptor may be further allowed to express another olfactory receptor. That is, a cell expressing a first olfactory receptor established by the above method or the like may be further allowed to express a second olfactory receptor. For example, a cell expressing a first olfactory receptor and a second olfactory receptor may be established by incorporating a gene encoding the second olfactory receptor into a vector and transfecting the constructed vector into a cell expressing the first olfactory receptor. A plurality of different olfactory receptors may be further expressed in a cell expressing the first olfactory receptor. FIG. 5 shows an example in which a vector containing a gene for Or-X, which is a second olfactory receptor, and a vector containing an antibiotic resistance gene are introduced into a cell expressing Or56a as the first olfactory receptor. FIG. 6 shows an example in which a vector containing both a gene for Or-X, which is a second olfactory receptor, and an antibiotic resistance gene are introduced into a cell expressing Or56a as the first olfactory receptor.

[0058] When arranging the odor detecting cells 20 in a single layer on the substrate 10 shown in FIG. 1, the odor detecting cells 20 may be fixed to the substrate 10 via a cell membrane modifying agent, as described above. For example, the substrate 10 is coated with a protein, and a solution containing the cell membrane modifying agent is dropped onto the protein covering the substrate 10, so that the protein covering the substrate 10 reacts with the substrate binding group of the cell membrane modifying agent, and the cell membrane modifying agent is bound to the protein covering the substrate 10. Thereafter, a solution containing the odor detecting cells 20 is dropped onto the substrate 10, so that the cell membrane binding group of the cell membrane modifying agent bound to the substrate 10 reacts with the odor detecting cells 20, and the odor detecting cells 20 are fixed to the substrate 10 via the cell membrane modifying agent.

[0059] Thereafter, the substrate 10 on which the odor detecting cells 20 are arranged is fixed in a tube 30 .

[0060] Next, an odor detection method according to an embodiment will be described. The odor detection method according to the embodiment includes contacting a fluid 40 to be subjected to an odor test with odor detection cells 20 arranged in a single layer on a substrate 10, the odor detection cells 20 having olfactory receptors and expressing fluorescent proteins whose fluorescence intensity changes depending on the concentration of odor molecules, and measuring the fluorescence emitted by the odor detection cells 20.

[0061] When the substrate 10 is fixed in the tube 30, the tube 30 may contain a buffer solution or a solution that appropriately contains a substance necessary for the survival of the odor detection cells 20. The odor detection cells 20 may be contacted with the odor test target fluid 40 by placing the odor test target fluid 40 in the tube 30. The odor test target fluid 40 is a liquid such as water.

[0062] For example, after placing the fluid 40 to be subjected to odor testing in the tube 30, the tube 30 is placed in a fluorometer and excitation light is irradiated onto the odor detection cells 20. As shown in Fig. 7, the odor detection cells 20 irradiated with the excitation light emit fluorescence with an intensity according to the concentration of odor molecules contained in the fluid 40 to be subjected to odor testing. When the fluorescent protein is GCaMP6s, the wavelength of the excitation light is, for example, 495 nm, and the wavelength of the fluorescence is 510 nm to 580 nm.

[0063] The odor of the fluid 40 being the subject of the odor test is evaluated based on the intensity of the fluorescence emitted by the odor detection cells 20. For example, the odor of the fluid 40 may be quantified based on the relationship between the intensity of the fluorescence previously acquired and the concentration of odor molecules or the strength of the odor. Alternatively, if the intensity of the fluorescence emitted by the odor detection cells 20 is equal to or greater than a preset reference value, the odor of the fluid 40 being the subject of the odor test may be evaluated as being equal to or greater than the reference value.

[0064] According to the odor detection method of this embodiment, it is possible to easily evaluate the odor of the fluid 40 to be subjected to odor testing by contacting the fluid 40 to be subjected to odor testing with odor detection cells 20 fixed in advance to a substrate 10.

[0065] In addition, in the odor detection method according to this embodiment, for example, if a portable tabletop fluorometer is used as the fluorometer, it is possible to quickly evaluate the odor of the fluid 40 to be subjected to odor testing at any location, not limited to a laboratory. For example, if the fluid 40 to be subjected to odor testing is water and the odor to be tested is a moldy odor, it is possible to collect water from an outdoor water source and test the water for a moldy odor on the spot.

[0066] (Example) (Example 1: Preparation of DmOr56a-expressing cells) The gene for the co-receptor DmOrco, derived from the antenna cDNA of Drosophila melanogaster, was amplified from the initiation codon to the termination codon with primers containing the following gene-specific sequences to obtain the DmOrco gene (full-length ORF). The obtained DmOrco gene was inserted into the multicloning site of the pIB vector (Invitrogen) to construct the pIB-DmOrco vector. DmOrco: Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGACAACCTCTATGCAACC-3' Reverse:5'-TTACCTTCGAACCGCTTACTTAAGCTGAACAAGAACCATGAAG-3'

[0067] Gene amplification by PCR was performed using forward and reverse primers at a concentration of 100 pmol / L each, PrimeSTAR HS DNA polymerase (Takara Bio Inc. R010A), the reaction buffer and dNTPs provided with the polymerase, according to the protocol provided with the polymerase. The temperature conditions for PCR were a step at 94°C for 2 minutes, followed by 30 cycles of 98°C for 10 seconds, 55°C for 10 seconds, and 72°C for 2 minutes, followed by a step at 72°C for 10 minutes.

[0068] Similarly, the DmOr56a gene (full-length ORF) was obtained by amplifying the gene from the start codon to the stop codon of the DmOr56a receptor gene derived from the antenna cDNA of Drosophila melanogaster using primers containing the following gene-specific sequences. The obtained DmOr56a gene was inserted into the multicloning site of the pIB vector to construct the pIB-DmOr56a vector. DmOr56a: Forward: 5'-CAGTGTGGTGGAATTGCCGCCATGTTTAAAGTTAAGGATCTGTTGC-3' Reverse:5'-GCCCTCTAGACTCGACTAATACAAGTGGGAGCTAC-3'

[0069] Next, the protein expression cassette of the pIB-DmOrco vector (the portion consisting of the OpIE2 promoter (P(OpIE2)), DmOrco, and OpIE2 polyA addition signal (OpIE2pA)) was amplified, and the amplified protein expression cassette was inserted into the Pci1 site of the pIB-DmOr56a vector to construct the olfactory receptor expression vector pIB-DmOr56a-DmOrco.

[0070] In addition, a calcium-sensitive protein (GCaMP6s) expression vector was constructed. The GCaMP6s gene was obtained from Dr. Douglas Kim (Janelia Farm Research Campus, Howard Hughes Medical Institute) via Addgene. The GCaMP6s gene was amplified from the initiation codon to the termination codon with primers containing the following gene-specific sequences to obtain the GCaMP6s gene (full-length ORF). The obtained GCaMP6s gene was inserted into the multicloning site of the pIZ vector (Invitrogen) to construct the pIZ-GCaMP6s vector. GCaMP6s: Forward: 5'-TTCGAATTTAAAGCTGCCGCCATGGGTTCTCATCATCATCATC-3' Reverse: 5'-TTACCTTCGAACCGCTCACTTCGCTGTCATCATTTGTAC-3'

[0071] The constructed olfactory receptor expression vector and calcium-sensitive protein expression vector were introduced into Sf21 cells by lipofection (TransIT-Insect; Mirus, registered trademark) according to the manual attached to TransIT-Insect. As a result, Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as the calcium-sensitive protein (GCaMP6s), were obtained. The DmOr56a receptor responds to moldy odor molecules.

[0072] (Example 2: Concentration dependence of response of DmOr56a-expressing cells) Geosmin, the causative substance of moldy odor, was serially diluted to 0 mol / L, 30 nmol / L, 100 nmol / L, 300 nmol / L, 1 μmol / L, 3 μmol / L, 10 μmol / L, and 30 μmol / L in assay buffer (140 mmol / L NaCl, 5.6 mmol / L KCl, 4.5 mmol / L CaCl2, 11.26 mmol / L MgCl2, 10 mmol / L HEPES, 9.4 mmol / L D-glucose, pH 7.2). The assay buffer and odor solutions for response measurement were all prepared to contain 0.1% DMSO at a final concentration. Next, each of the diluted solutions was contacted with Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s. As a result, as shown in FIG. 8, Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s, emitted fluorescence with an intensity that depended on the concentration of geosmin.

[0073] (Example 3: Odor molecule specificity of responses of DmOr56a-expressing cells) A solution containing linalool at a concentration of 10 μmol / L, a solution containing decanal at a concentration of 10 μmol / L, a solution containing octanal at a concentration of 10 μmol / L, a solution containing citral at a concentration of 10 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L were prepared. Linalool is an odor molecule that gives off lily of the valley, lavender, and bergamot-like scents. Decanal is an odor molecule that gives off citrus scents. Octanal is an odor molecule that gives off fruity scents. Citral is an odor molecule that gives off lemon scents.

[0074] A solution containing each odor molecule was contacted with Sf21 cells expressing the DmOr56a receptor, its co-receptor DmOrco, and GCaMP6s. As a result, as shown in Figure 9, Sf21 cells expressing the DmOr56a receptor, its co-receptor DmOrco, and GCaMP6s emitted fluorescence in response to geosmin specifically.

[0075] (Example 4: Detection of odor molecules using a suspension of DmOr56a-expressing cells) Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s, were cultured as adherent cells in a T-25 flask. The cells were then scraped off from the T-25 flask with a scraper (IWAKI; 9020-250) and the cell suspension was transferred to a 50 mL tube. The tube was centrifuged at 500 × g, and the supernatant was removed from the tube.

[0076] 20 mL of assay buffer was added to the tube, and the cell pellet was suspended in the assay buffer. The tube was then centrifuged at 500 × g. After centrifugation, the supernatant was removed from the tube, and 1 × 10 7 An assay buffer was added to the tube so as to give a concentration of cells / mL, and the cells were suspended to obtain a cell suspension.

[0077] 180 μL of the cell suspension was placed in a 0.5 mL tube (Promega; E4941), and the tube was placed in a fluorometer (Quantus, Fluorometer, registered trademark) to measure the basal fluorescence intensity as shown in FIG. 10. Pipetting and fluorescence measurement were repeated for 5 seconds until 10 consecutive measurements of fluorescence intensity with a fluctuation of ±2% were obtained, and the basal fluorescence intensity was stabilized. After the basal fluorescence intensity was stabilized, 20 μL of 10 μmol / L geosmin was dropped into the cell suspension in the tube. After the drop, the fluorescence intensity was measured every 20 seconds. As a result, it was confirmed that the cells emitted fluorescence in response to geosmin. However, it was shown that it took a long time for the basal fluorescence intensity to stabilize unless the cells were fixed.

[0078] Example 5: Establishment of homogeneous odor-detecting cell lines A subculture medium was prepared by adding 10% US Insect Cell Screened FBS (GE Healthcare; SH30070.03) and three types of antibiotics (10 μg / mL Gentamicin Reagent Solution (Gibco; 15710-064), 10 μg / mL Blasticidin S HCl (Gibco; A11139-03), and 100 μg / mL Zeocin (Invitrogen; R25001)) to Grace's Insect Medium, Supplemented (Gibco; 11605-094). Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s, were subcultured in a flask (FALCON; 353082) using the subculture medium. The volume of the cell suspension at the time of subculture was 6 mL. When the cells became confluent, 6 mL of the supernatant was collected from the flask and placed in a 15 mL tube (TPP; 91015). The 15 mL tube was centrifuged at 400×g and 4° C. for 3 minutes using a microcentrifuge.

[0079] After centrifugation, the supernatant was sterilized using a 10 mL syringe (TOP; 01007) and a 0.45 μm filter (CORNING; 431220). The sterilized supernatant was mixed with an equal volume of fresh subculture medium containing antibiotics (10 μg / mL Blasticidin S HCl, 100 μg / mL Zeocin) to prepare 10 mL of conditioned medium.

[0080] The cells attached to the bottom of the flask from which the supernatant was collected were detached and suspended in 1 mL of new medium, and the cell suspension was collected in a 1.5 mL tube (AXYGEN; MCT-150-C). A cell suspension containing 40 cells was extracted, added to the above-mentioned conditioned medium, and pipetted thoroughly. The entire amount of the conditioned medium to which the cells had been added was transferred to a reservoir (BMBio; BM-0850-1). Furthermore, 100 μL of the conditioned medium to which the cells had been added was dropped into a 96-well plate (IWAKI; 3860-096) using an 8-channel pipette (HTL; HT5123), and the cells were then cultured at 27 °C. After the cells had adhered to the wells, the wells were observed with an inverted microscope to confirm that only single cells were present in the conditioned medium.

[0081] The cells in the wells where single cells were confirmed at the time of seeding were cultured until they reached approximately 80% to 90% confluence. The cells were then scaled up in the following order: 24-well plates (IWAKI; 3820-024), 35 mm dishes (CORNING; 353801), and T-25 flasks. The amount of medium was adjusted to 500 μL, 2.5 mL, and 5 mL, respectively, for culture in the 24-well plates, 35 mm dishes, and T-25 flasks, and the cells were cultured at 27°C. The responsiveness of the cells that could be scaled up to the T-25 flask was investigated by calcium imaging, and the cell line that showed good responsiveness was obtained as a homogenous odor detection cell line.

[0082] Example 6: Reactivity of homogeneous odor-detecting cell lines The fluorescence responses to odorants of non-homogenized Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, and GCaMP6s, and of homogenized Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, and GCaMP6s, were measured using the following procedure.

[0083] After seeding and adhering the cells on a 12 mm diameter cover glass (CS-12R: Warner Instruments, LLC), the cover glass was inserted into an open bath chamber for circular cover glass (RC-48LP: Warner Instruments, LLC). To perfuse the solution in the chamber and add odor molecules, silicone tubes with an inner diameter of 1 mm and an outer diameter of 3 mm connected to a peristaltic tube pump (MP-2010: Tokyo Rikakikai Co. Ltd.) were placed at the inlet and outlet of the chamber using a homemade holder. The perfusion conditions were a flow rate of approximately 1.5 mL / min, the volume of the solution in the chamber was approximately 200 μL, and the odor stimulation time to the cells was 15 seconds with 10 μmol / L geosmin.

[0084] The fluorescence response of the cells in the chamber was measured using a microscope. The microscope used was an upright fluorescence microscope (BX51WI: Olympus) equipped with a 20x water immersion objective lens (UMPlanFI 20x / 0.50 W: Olympus). An EM-CCD camera (DU-897E: Andor Technology PLC) was used to measure the changes in the fluorescence intensity of the cells. The camera was operated with AndoriQ (Andor Technology) to acquire images of 512 x 512 pixels. The microscope filter used was a fluorescent filter set for GFP (U-MGFPHQ: Olympus). A 100W halogen lamp (TH4-100, Olympus) was used as the light source, and the exposure time for fluorescence observation was set to 500 milliseconds. Images were taken every second.

[0085] Figure 11 shows a graph showing the relationship between the rate of change in fluorescence intensity before and after the application of odor molecules and the percentage of cell counts. When a non-homogenized cell line was used, 67.7% of the total cells had a fluorescence response of 5% or more. Furthermore, many cells had a low rate of fluorescence change when odor molecules were applied, with an average rate of change of 26.7%. In contrast, when a homogenized cell line was used, 92.0% of the total cells had a fluorescence response. Furthermore, many cells had a high rate of fluorescence change when odor molecules were applied, with an average rate of change of 45.9%. Therefore, it was demonstrated that odor molecules can be detected with high sensitivity when a homogenized cell line is used.

[0086] Example 7: Long-term stability of odor-detecting cells A homogenized cell line with a fluorescence change rate of 36.1% upon odor stimulation immediately after cloning was passaged for a long period of time, and the time-dependent change in the fluorescence change rate upon odor stimulation was examined. As a result, as shown in FIG. 12, the fluorescence change rate in response to odor stimulation was equal to or greater than that immediately after cloning for three and a half months after cloning. In addition, the homogenized cell line was frozen and stored for three and a half months immediately after cloning, and after thawing, the cell line was passaged for a long period of time, and the time-dependent change in the fluorescence change rate upon odor stimulation was examined. As a result, as shown in FIG. 13, the fluorescence change rate in response to odor stimulation was equal to or greater than that immediately after cloning for two months after thawing. In addition, the homogenized cell line was frozen and stored for six and a half months immediately after cloning, and after thawing, the cell line was passaged for a long period of time, and the time-dependent change in the fluorescence change rate upon odor stimulation was examined. As a result, as shown in FIG. 14, the fluorescence change rate in response to odor stimulation was equal to or greater than that immediately after cloning for one and a half months after thawing.

[0087] (Example 8: Preparation of odor detection kit) A 76mm x 26mm slide glass (MATSUNAMI, S9441) was cut into 4mm x 26mm pieces using a glass cutting diamond cutter (TRUSCO, 419-9847). Approximately 100μL of collagen solution was dropped onto the cut glass, and the slide glass was left to stand in a clean bench for 30 minutes. The collagen solution used was Cellmatrix Type I-C (Nitta Gelatin Co., Ltd., 63100771) diluted 10 times with HCl (FUJIFILM Wako Pure Chemical Co., Ltd., pH = 3).

[0088] After 30 minutes, excess collagen solution was removed from the slide glass, and an appropriate amount of 100 μmol / L cell membrane modifier (oleyl-PEG-NHS) solution was dropped onto the slide glass and left to stand at 37 ° C for 30 minutes. The cell membrane modifier solution was prepared by diluting a mixture of 0.4 mg of SUNBRIGHT (NOF, OE-040CS) and 10 mL of ultra-dehydrated DMSO 100 times with D-PBS (Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795). After 30 minutes, excess cell membrane modifier solution was removed from the slide glass and left to stand for 20 minutes.

[0089] After 20 minutes, about 1×10 7 A suspension of homogenized odor detection cells (solvent: assay buffer) was dropped onto the surface of a slide glass treated with a cell membrane modifying agent and allowed to stand for 10 minutes. After that, the slide glass with the odor detection cells fixed in a monolayer was fixed in a 0.5 mL tube filled with 400 μL of assay buffer containing 0.1% DMSO, thereby producing an odor detection kit according to the embodiment.

[0090] (Example 9: Odor measurement using a kit and a fluorometer) Immediately after preparing the odor detection kit according to the embodiment, in order to confirm that the base fluorescence intensity was stable, the odor detection kit according to the embodiment was placed in a fluorometer (Quantus, Fluorometer, registered trademark) and waited until 10 points where the range of the rate of change in the fluorescence intensity was within ±2% could be measured. Next, 200 μL of 10 μmol / L geosmin was added to 400 μL of assay buffer in the tube of the odor detection kit according to the embodiment, and the solution in the tube was pipetted for 10 seconds. After that, the rate of change in the fluorescence intensity was measured every 20 seconds for 140 seconds. As a result, the fluorescence response to geosmin was measured as shown in FIG. 15. When the same measurement was repeated using a plurality of odor detection kits according to the embodiment, the fluorescence response to geosmin was stably measured in all cases. Note that when the slide glass was not fixed in the tube, the base fluorescence intensity was not stable, and the fluorescence response to geosmin could not be stably measured.

[0091] (Example 10: Detection of low concentration odor molecules using a kit and a fluorometer) Geosmin was added to the odor detection kits according to the examples to final concentrations of 0 mol / L, 10 pmol / L, 100 pmol / L, 1 nmol / L, 10 nmol / L, 100 nmol / L, 1 μmol / L, and 10 μmol / L, respectively, and the rate of change in fluorescence intensity was measured using a fluorometer (Quantus, Fluorometer, registered trademark). The results are shown in FIG. The rate of change in fluorescence intensity when the geosmin concentration was 0 mol / L was 1.5 ± 0.4%, when the geosmin concentration was 10 pmol / L was 2.7 ± 0.5%, when the geosmin concentration was 100 pmol / L was 5.5 ± 1.0%, when the geosmin concentration was 1 nmol / L was 8.1 ± 0.8%, when the geosmin concentration was 10 nmol / L was 12.0 ± 1.0%, when the geosmin concentration was 100 nmol / L was 21.3 ± 2.0%, when the geosmin concentration was 1 μmol / L was 35.0 ± 5.3%, and when the geosmin concentration was 10 μmol / L was 29.4 ± 4.3%, indicating that the fluorescence intensity increased depending on the geosmin concentration.

[0092] The rate of change in the measured fluorescence intensity was examined using the significance level obtained by correcting the p-value calculated by the Welch's t-test using the Holm method. As a result, a significant difference was observed for geosmin at 100 pmol / L or more compared to geosmin at 0 mol / L. Therefore, it was shown that the odor detection kit and fluorometer according to the embodiment can detect odor molecules with the same sensitivity as GC / MS. For example, the concentration of geosmin in a dam lake that is the source of tap water in Japan was 33.5 nmol / L or less. Therefore, it was shown that the odor detection kit and fluorometer according to the embodiment can detect the mold odor of water collected from the dam lake. In addition, the lower limit of the human sensory evaluation of the geosmin solution prepared as described above was about 10 nmol / L. Therefore, it was shown that the odor detection kit and fluorometer according to the embodiment can detect low-concentration mold odors that cannot be detected by human sensory evaluation.

[0093] (Example 11: Specific detection of odor molecules using a kit and a fluorometer) In the dam lake water, multiple types of mold odors such as geosmin and 2-methylisoborneol (2-MIB) are generated. Therefore, in order to evaluate the mold odor selectivity of the odor detection kit according to the embodiment, 10 μmol / L of geosmin, 10 μmol / L of 2-MIB, and an assay buffer (control) containing neither mold odor were dropped into the odor detection kit according to the embodiment, and the rate of change in fluorescence intensity was measured using a fluorometer (Quantus, Fluorometer, registered trademark).

[0094] As a result, as shown in Figure 17, the rate of change in fluorescence intensity when the control was dropped was 0.1 ± 0.6%, the rate of change in fluorescence intensity when 2-MIB was dropped was -0.3 ± 1.1%, and the rate of change in fluorescence intensity when geosmin was dropped was 13.7 ± 3.2%. The measured rate of change in fluorescence intensity was examined using a significance level obtained by correcting the p-value calculated by Welch's t-test using the Holm method. As a result, a significant difference was observed with 10 μmol / L geosmin compared to the control, but no significant difference was observed with 10 μmol / L 2-MIB. Therefore, it was demonstrated that the odor detection kit according to the embodiment prepared specifically detects geosmin.

[0095] Example 12: Comparison of stability of cell suspension and kit A suspension of odor detection cells to which no odor molecules were added was placed in a fluorometer (Quantus, Fluorometer, registered trademark), and the time until the rate of change in the base fluorescence intensity stabilized at ±2% was compared with the time until the rate of change in the base fluorescence intensity stabilized at ±2% when an odor detection kit according to an embodiment to which no odor molecules were added was placed in the fluorometer. As a result, as shown in FIG. 18, the time until stability was reached was 300±30 seconds for the suspension of odor detection cells, and 70±6 seconds for the odor detection kit according to the embodiment. This indicates that the odor detection kit according to the embodiment in which odor detection cells are arranged in a single layer on a substrate takes a shorter time to stabilize.

[0096] (Example 13: Comparison of responsiveness between cell suspension and kit) In order to compare the response of the suspension of odor detection cells to odor molecules with the response of the odor detection kit according to the embodiment to odor molecules, an assay buffer (control) and 10 μmol / L of geosmin were added to each, and the rate of change in fluorescence intensity after 120 seconds was measured using a fluorometer (Quantus, Fluorometer, registered trademark). As a result, as shown in FIG. 19, in the case of the suspension of odor detection cells, the rate of change in fluorescence intensity after the addition of the control was -3.9±6.5%, and the rate of change in fluorescence intensity after the addition of geosmin was 3.2±3.6%. In the case of the odor detection kit according to the embodiment, the rate of change in fluorescence intensity after the addition of the control was 2.4±1.7%, and the rate of change in fluorescence intensity after the addition of geosmin was 27.5±4.3%. Thus, it was shown that when the odor detection kit according to the embodiment was used, the rate of change in fluorescence intensity when the control was added was small, and the rate of change in fluorescence intensity when the odor molecules were added was large.

[0097] (Example 14: Comparison of variability in measurement results between cell suspension and kit) Based on the results shown in FIG. 19, the absolute value of the coefficient of variation of the rate of change in each fluorescence intensity was calculated. The absolute value of the coefficient of variation represents the variation. As a result, as shown in FIG. 20, in the case of the suspension of odor detection cells, the absolute value of the coefficient of variation of the rate of change in fluorescence intensity after the addition of the control was 165.9%, and the absolute value of the coefficient of variation of the rate of change in fluorescence intensity after the addition of geosmin was 113.8%. In the case of the odor detection kit according to the embodiment, the absolute value of the coefficient of variation of the rate of change in fluorescence intensity after the addition of the control was 71.2%, and the absolute value of the coefficient of variation of the rate of change in fluorescence intensity after the addition of geosmin was 15.5%. This shows that the measurement results have less variation when the odor detection kit according to the embodiment is used.

[0098] (Example 15: Measurement of water obtained from a dam lake) At the shore of a dam lake, the odor detection kit according to the embodiment and a fluorometer (Quantus, Fluorometer, registered trademark) were used to measure the rate of change in fluorescence intensity when the assay buffer alone (control) was added to the kit and when the assay buffer containing 100 μmol / L of geosmin was added to the kit. As a result, as shown in FIG. 21, when the assay buffer alone (control) was added to the kit, the rate of change in fluorescence intensity was 2.8%, and when the assay buffer containing 100 μmol / L of geosmin was added to the kit, the rate of change in fluorescence intensity was 21.2%. This shows that the odor detection kit according to the embodiment can detect odor molecules even outdoors.

[0099] Next, at the shore of a dam lake, the odor detection kit according to the embodiment and a fluorometer (Quantus, Fluorometer, registered trademark) were used to measure the rate of change in fluorescence intensity when the surface water of the dam water was added to the kit and when the surface water of the dam water to which 100 μmol / L of geosmin had been added was added to the kit. As a result, as shown in FIG. 21, when only the surface water of the dam water was added to the kit, the rate of change in fluorescence intensity was 6.0%, and when the surface water of the dam water to which 100 μmol / L of geosmin had been added was added to the kit, the rate of change in fluorescence intensity was 13.7%. When the surface water of the dam water was analyzed by GC / MS, it was found to contain 300 pmol / L of geosmin. Therefore, it was shown that the odor detection kit according to the embodiment can detect specific odor molecules even when the dam lake water containing background odors and impurities is used as the test target.

[0100] (Example 16: Preparation of cells expressing DmOr56a and DmOr82a receptors) Using the method described in Example 5, a homogeneous odor-detecting cell line was generated from Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s.

[0101] In addition, the gene for the receptor DmOr82a, derived from the antenna cDNA of Drosophila melanogaster, was amplified from the start codon to the stop codon using primers containing the following gene-specific sequences, and inserted into the multicloning site (BamHI, NotI) of the pIEx4 vector (Novagen) to construct the pIEx4-DmOr82a vector. DmOr82a Forward; 5'-ACGTCAAGAGCTCGCGGATCGCCGCCATGGGTAGGCTGTTTCAACTG-3' Reverse; 5'-CTTTACCAGAAGATGCGGCCTTACTCAATAGATTTGATGAG-3'

[0102] The constructed pIEx4-DmOr82a vector was transfected into a homogeneous odor-detecting cell line expressing the DmOr56a receptor together with the pIE1-neo vector (Novagen; containing a G418 resistance gene) by lipofection (TransIT-insect; Mirus, registered trademark) according to the manual attached to the TransIT-Insect. This produced cells expressing the DmOr82a receptor in addition to the DmOr56a receptor.

[0103] (Example 17: Preparation of cells expressing DmOr56a and DmOr49b receptors) Using the method described in Example 5, a homogeneous odor-detecting cell line was generated from Sf21 cells expressing the DmOr56a receptor and co-receptor DmOrco, as well as GCaMP6s.

[0104] In addition, the G418 resistance gene derived from the pIE1-neo vector was amplified from the initiation codon to the termination codon using primers containing the following gene-specific sequences, and inserted into the multicloning site (HindIII, SacII) of the pIZ vector (pIZ / V5-His (Invitrogen) to construct the pIZ-neo vector. G418(neo) Forward; 5'-ATCTGTTCGAATTTAAAGCTGCCGCCATGATTGAACAAGATGGATTGC-3' Reverse; 5'-GATAGGCTTACCTTCGAACCTCAGAAGAACTCGTCAAGAAG-3'

[0105] Next, the protein expression cassette of the pIZ-neo vector (the portion connected to the OpIE2 promoter (P(OpIE2)), neo gene, and OpIE2 polyA addition signal (OpIE2pA)) was amplified, and the amplified protein expression cassette was inserted into the Pci1 site of the pIB vector to construct the G418 resistance gene insertion vector pIB-neo vector.

[0106] Next, the gene for the receptor DmOr49b, derived from the antenna cDNA of Drosophila melanogaster, was amplified from the start codon to the stop codon using primers containing the following gene-specific sequences, and inserted into the multicloning site (EcoRI, XhoI) of the pIB-neo vector (above) to construct the pIB-DmOr49b-neo vector. DmOr49b Forward; 5'-CAGTGTGGTGGAATTGCCGCCATGTTTGAAGACATTCAGCTAATC-3' Reverse; 5'-GCCCTCTAGACTCGATCATCCGTAGACTCGCTTG-3'

[0107] The constructed pIB-DmOr49b-neo vector was transfected into a homogeneous odor-detecting cell line expressing the DmOr56a receptor by lipofection (TransIT-insect; Mirus, registered trademark) according to the manual attached to the TransIT-Insect. This produced cells expressing the DmOr49b receptor in addition to the DmOr56a receptor.

[0108] (Example 18: Reactivity of DmOr56a receptor and DmOr82a receptor expressing cells) Cells expressing DmOr56a and DmOr82a receptors were generated using the method described in Example 16.

[0109] In addition, a solution containing no odor (negative control), a solution containing geranyl acetate at a concentration of 300 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L were prepared. Geranyl acetate is the target odor of the DmOr82a receptor. Geosmin is the target odor of the DmOr56a receptor. As shown in Figure 22, the negative control solution, the solution containing geranyl acetate, and the solution containing geosmin were contacted with cells expressing the DmOr56a receptor and the DmOr82a receptor in that order. As a result, the fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr82a receptor changed by 23.2% in response to geranyl acetate and 16.4% in response to geosmin.

[0110] (Example 19: Reactivity of cells expressing DmOr56a and DmOr49b receptors) Cells expressing DmOr56a and DmOr49b receptors were generated using the method described in Example 17.

[0111] In addition, a solution containing no odor (negative control), a solution containing 2-methylphenol at a concentration of 300 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L were prepared. 2-Methylphenol is the target odor of the DmOr49b receptor. Geosmin is the target odor of the DmOr56a receptor. As shown in Figure 23, the negative control solution, the solution containing 2-methylphenol, and the solution containing geosmin were contacted with cells expressing the DmOr56a receptor and the DmOr49b receptor in that order. As a result, the fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr49b receptor changed by 37.6% in response to 2-methylphenol and by 29.4% in response to geosmin.

[0112] (Example 20: Concentration-dependent reactivity of DmOr56a receptor and DmOr82a receptor expressing cells) Cells expressing DmOr56a and DmOr82a receptors were generated using the method described in Example 16.

[0113] Geranyl acetate was serially diluted with assay buffer (140mmol / L NaCl, 5.6mmol / L KCl, 4.5mmol / L CaCl2, 11.26mmol / L MgCl2, 10mmol / L HEPES, 9.4mmol / L D-glucose, pH 7.2) to final concentrations of 0mol / L (control), 1μmol / L, 3μmol / L, 10μmol / L, 30μmol / L, 100μmol / L, and 300μmol / L. Geosmin was diluted in assay buffer as a positive control to a final concentration of 10μmol / L. All solutions were prepared to contain DMSO at a final concentration of 0.1%.

[0114] Next, as shown in Figure 24, each solution was contacted with cells expressing DmOr56a and DmOr82a receptors at 6-minute intervals. As a result, as shown in Figures 24 and 25, cells expressing DmOr56a and DmOr82a receptors emitted fluorescence with an intensity that depended on the concentration of geranyl acetate. They also emitted fluorescence in response to geosmin, which was a positive control.

[0115] (Example 21: Concentration-dependent reactivity of DmOr56a receptor and DmOr49b receptor expressing cells) Cells expressing DmOr56a and DmOr49b receptors were generated using the method described in Example 17.

[0116] 2-Methylphenol was serially diluted with the assay buffer to final concentrations of 0 mol / L (control), 1 μmol / L, 3 μmol / L, 10 μmol / L, 30 μmol / L, 100 μmol / L, and 300 μmol / L. As a positive control, geosmin was diluted with the assay buffer to a final concentration of 10 μmol / L. All solutions were prepared to contain DMSO at a final concentration of 0.1%.

[0117] Next, as shown in Figure 26, each solution was contacted with cells expressing DmOr56a and DmOr49b receptors at 6-minute intervals. As a result, as shown in Figures 26 and 27, cells expressing DmOr56a and DmOr49b receptors emitted fluorescence with an intensity that depended on the concentration of 2-methylphenol. They also emitted fluorescence in response to geosmin, which is a positive control.

[0118] (Example 22: Specific reactivity of olfactory receptor expressing cells) Cells expressing the DmOr56a receptor were prepared using the method described in Example 5, cells expressing the DmOr56a receptor and the DmOr82a receptor were prepared using the method described in Example 16, and cells expressing the DmOr56a receptor and the DmOr49b receptor were prepared using the method described in Example 17.

[0119] In addition, we prepared a solution containing no odor (negative control), a solution containing benzaldehyde at a concentration of 300 μmol / L, a solution containing methyl benzoate at a concentration of 300 μmol / L, a solution containing 1-octen-3-ol at a concentration of 300 μmol / L, a solution containing 2-methylphenol at a concentration of 300 μmol / L, a solution containing geranyl acetate at a concentration of 300 μmol / L, and a solution containing geosmin at a concentration of 10 μmol / L.

[0120] When each solution was contacted with cells expressing the DmOr56a receptor, the fluorescence intensity of the cells expressing the DmOr56a receptor changed by 68.1% in response to geosmin, as shown in Figure 28. The fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr82a receptor changed by 7.4% in response to benzaldehyde, 34.5% in response to geranyl acetate, and 4.3% in response to geosmin. The fluorescence intensity of the cells expressing the DmOr56a receptor and the DmOr49b receptor changed by 19.4% in response to benzaldehyde, 58.5% in response to 2-methylphenol, and 29.6% in response to geosmin. [Explanation of symbols]

[0121] 10 Substrate, 20 Odor-detecting cell, 30 Tube, 40 Fluid

Claims

1. A substrate; Odor detection cells arranged in a single layer on the substrate, the cells having olfactory receptors and expressing fluorescent proteins whose fluorescence intensity changes depending on the concentration of odor molecules; a tube in which the substrate can be fixed, the tube being for containing a liquid to be subjected to odor testing, the tube being capable of being installed in a fluorometer; Equipped with The inner diameter of the tube and the width of the substrate are the same; Odor detection kit for fluorometer.

2. The odor detection kit according to claim 1 , wherein the odor detection cells are fixed to the substrate via a cell membrane modifying agent.

3. The odor detection kit according to claim 1 or 2, wherein the odor detection cells express an olfactory receptor by an introduced gene.

4. The odor detection kit according to claim 1 , wherein the odor detection cells express an insect olfactory receptor.

5. The odor detection kit according to claim 1 , wherein the olfactory receptor is an ionotropic receptor.

6. The odor detection kit according to claim 5 , wherein the fluorescent protein changes the fluorescence intensity depending on an ion concentration.

7. The odor detection kit according to any one of claims 1 to 6, wherein the olfactory receptor is selected from BmOR1, BmOR3, DmOr13a, DmOr56a, DmOr82a, DmOr49b, DmOr85b and PxOR1.

8. The odor detection kit according to claim 1 , wherein the odor detection cells are insect cells.

9. The odor detection kit according to claim 8 , wherein the insect cells are cells derived from a moth.

10. The odor detection kit according to claim 8 or 9, wherein the insect cells are selected from Sf21, Sf9, High Five, and Tni derived cells.

11. The odor detection kit according to claim 8 , wherein the insect cells are cells derived from Drosophila melanogaster.

12. The odor detection kit of claim 8 , wherein the insect cells are Drosophila S2 cells.

13. The odor detection kit according to claim 1 , wherein the substrate is transparent.

14. preparing an odor detection kit for a fluorometer, the kit comprising: a substrate; odor detection cells arranged in a single layer on the substrate, the odor detection cells having olfactory receptors and expressing fluorescent proteins whose fluorescence intensity changes depending on the concentration of odor molecules; and a tube in which the substrate can be fixed and which can be installed in a fluorometer; Putting a liquid to be odor-tested into the tube; placing the tube in the fluorometer and irradiating the odor detection cells with excitation light; Including, The inner diameter of the tube and the width of the substrate are the same; Odor detection methods.

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