CN118308457B - Use of olfactory receptors for the recognition of trimethylamine and method for detecting trimethylamine - Google Patents
Use of olfactory receptors for the recognition of trimethylamine and method for detecting trimethylamine Download PDFInfo
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Abstract
The application belongs to the technical field of chemical detection, and particularly discloses application of an olfactory receptor in trimethylamine recognition and a method for detecting trimethylamine. Use of the olfactory receptor rTAAR8c for the recognition of trimethylamine. The method comprises the following steps: contacting a sample to be tested with an olfactory receptor rTAAR8c, and determining a response value of the olfactory receptor rTAAR8c after contact; and determining whether trimethylamine is contained in the sample to be detected based on the response value. According to the application, trimethylamine can activate an olfactory receptor rTAAR8c, so that a sample to be detected is contacted with the olfactory receptor rTAAR8c, if the sample to be detected contains trimethylamine, the olfactory receptor rTAAR8c can be activated, a response value of the olfactory receptor rTAAR8c after activation is obtained, and whether the sample to be detected contains trimethylamine can be determined according to the response value.
Description
Technical Field
The application belongs to the technical field of chemical detection, and particularly relates to application of an olfactory receptor in recognizing trimethylamine and a method for detecting trimethylamine, and more particularly relates to application of the olfactory receptor in recognizing trimethylamine, application of the trimethylamine in activating the olfactory receptor, a method for detecting trimethylamine, a method for detecting freshness of meat products, a detection device, electronic equipment and an executable storage medium.
Background
Trimethylamine (chemical formula C 3H9 N) is an organic compound. It is a colorless gas at normal temperature and pressure, has fish oil smell, and can be dissolved in water, ethanol and diethyl ether. In addition, it can react with oxidant, anhydride and mercury, and has corrosiveness to metals such as aluminum, magnesium, zinc, tin, copper and copper alloy.
Trimethylamine is widely used mainly as a disinfectant, an alarm agent for natural gas, an analytical reagent and an organic synthetic raw material, and is also used as a raw material for medicines, agricultural chemicals, photographic materials, rubber additives, explosives, chemical fiber solvents, surfactants and dyes. Trimethylamine can also be used as a disinfectant and analytical reagent in food products, where it can be used in certain specific process steps, such as sterilization and raw material preparation, during food processing and manufacturing. In addition, trimethylamine is associated with the presence of ingredients in food products. For example, when a fish dies, trimethylamine is degraded, which is also responsible for the fishy smell of the fish.
Therefore, the detection of trimethylamine is of significance, and development of a novel method for detecting trimethylamine is highly demanded.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the application provides the use of an olfactory receptor for the recognition of trimethylamine and a method for detecting trimethylamine.
The present application has been completed based on the following findings by the inventors:
Mammals have excellent olfactory perceptibility and can sensitively and rapidly identify and distinguish odors in foods. During the course of the study, olfactory receptors were screened from animal olfactory receptors (Odorant receptors (ORs)) for compounds that respond to the corresponding compounds of different olfactory receptors and were used to aid in the detection of compounds under various conditions.
Based on this, the present inventors screened olfactory receptors responding to trimethylamine using olfactory receptors of human, mouse, rat or zebra fish, and found that hTAAR5, mTAAR5 and rTAAR8c can effectively recognize trimethylamine during screening, and that rTAAR8c has higher response intensity, sensitivity and accuracy than hTAAR5 and mTAAR 5.
In a first aspect of the application, the application proposes the use of the olfactory receptor rTAAR8c for the recognition of trimethylamine. The application adopts trimethylamine to stimulate the olfactory receptor rTAAR8c, and then can effectively activate rTAAR8c, so that the application adopts the olfactory receptor rTAAR8c to identify the trimethylamine, can be used for detecting whether the sample contains the trimethylamine, and lays a foundation for the identification of the trimethylamine and the detection of the sample containing the trimethylamine.
According to an embodiment of the application, said recognition is manifested by a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
In a second aspect of the application, the application proposes the use of trimethylamine for activating the olfactory receptor rTAAR8 c. The application adopts trimethylamine to stimulate the olfactory receptor rTAAR8c, and then can effectively activate rTAAR8c, so that the application adopts the olfactory receptor rTAAR8c to identify the trimethylamine, can be used for detecting whether the sample contains the trimethylamine, and lays a foundation for the identification of the trimethylamine and the detection of the sample containing the trimethylamine.
According to an embodiment of the application, the activation is manifested by a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
In a third aspect of the application, the application provides a method for detecting trimethylamine. According to an embodiment of the application, the method comprises: contacting a sample to be tested with an olfactory receptor rTAAR8c, and determining a response value of the olfactory receptor rTAAR8c after contact; and determining whether trimethylamine is contained in the sample to be detected based on the response value. From the foregoing, it can be seen that trimethylamine activates the olfactory receptor rTAAR8c, and thus, the sample to be tested is contacted with the olfactory receptor rTAAR8c, if trimethylamine is contained in the sample to be tested, the olfactory receptor rTAAR8c can be activated, and a response value of the olfactory receptor rTAAR8c after activation is obtained, and whether trimethylamine is contained in the sample to be tested can be determined according to the response value.
According to an embodiment of the present application, the presence response value of the olfactory receptor rTAAR8c is an indication that trimethylamine is contained in the sample to be tested, or the absence response value of the olfactory receptor rTAAR8c is an indication that trimethylamine is not contained in the sample to be tested.
According to an embodiment of the application, the method further comprises: and determining the content of trimethylamine in the sample to be detected based on a standard curve, wherein the standard curve is a corresponding curve of a preset amount of trimethylamine and an olfactory receptor rTAAR8c response value.
According to an embodiment of the application, the olfactory receptor rTAAR8c is provided by a cell or transgenic cell expressing the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the concentration of Ca 2+ in the cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of olfactory receptor protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
In a fourth aspect of the present application, a method of detecting freshness of a meat product is provided. According to an embodiment of the application, the method comprises: contacting meat food to be detected with an olfactory receptor rTAAR8c, and determining a response value of the olfactory receptor rTAAR8c after contact; and determining the freshness of the meat food to be tested based on the response value. From the foregoing, it can be seen that trimethylamine can activate the olfactory receptor rTAAR8c, and thus, the sample to be tested is contacted with the olfactory receptor rTAAR8c, if trimethylamine is contained in the sample to be tested, the olfactory receptor rTAAR8c can be activated, a response value of the olfactory receptor rTAAR8c after activation is obtained, trimethylamine is contained in the food to be tested can be determined based on the response value, and the freshness of the meat food to be tested can be judged according to the trimethylamine.
According to an embodiment of the application, the olfactory receptor rTAAR8c presence response value is an indication that the meat product under test is not fresh, or the olfactory receptor rTAAR8c absence response value is an indication that the meat product under test is fresh.
According to an embodiment of the application, the method further comprises: and determining the content of trimethylamine in the meat food to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the response value of the predetermined amount of trimethylamine and the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the olfactory receptor rTAAR8c is provided by a cell or transgenic cell expressing the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the concentration of Ca 2+ in the cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
In a fifth aspect of the application, the application proposes a detection device for detecting freshness of meat products and/or for detecting trimethylamine. According to an embodiment of the present application, the detection device includes: a detection unit for acquiring a response value of an olfactory receptor rTAAR8c to a sample to be tested, wherein the detection unit contains the olfactory receptor rTAAR8c; and the determining unit is used for determining whether trimethylamine is contained in the sample to be detected or whether the sample to be detected is fresh or not based on the response value. Therefore, whether the sample to be detected contains trimethylamine or not can be detected rapidly and accurately, or whether the sample to be detected (such as meat sample) is fresh or not can be detected rapidly and accurately.
In a sixth aspect of the application, the application provides an electronic device. According to an embodiment of the present application, the electronic device includes: a memory and a processor; the memory is used for storing a computer program; the processor is configured to execute the computer program to implement the method according to the third or fourth aspect. The electronic equipment can be used for detecting whether the sample to be detected contains trimethylamine or not or determining whether the sample to be detected (such as a meat sample) is fresh or not, and has the advantages of simplicity and convenience in operation, rapidness in identification, high accuracy and the like.
In a seventh aspect of the present application, the present application proposes an executable storage medium. According to an embodiment of the application, the storage medium stores computer program instructions which, when run on a processor, cause the processor to perform the method according to the third or fourth aspect.
Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application.
Drawings
The foregoing and/or additional aspects and advantages of the application will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the response of trimethylamine to stimulate the olfactory TAAR receptor library of human, mouse, rat or zebra fish in example 1 of the present application;
FIG. 2 is a graph showing the dose dependence of hTAAR5, mTAAR5 and rTAAR8c in response to trimethylamine in example 2 of the present application;
FIG. 3 is a graph showing the real-time response of hTAAR5, mTAAR5 and rTAAR8c to trimethylamine in example 2 of the present application.
Detailed Description
Embodiments of the present application are described in detail below. The following examples are illustrative only and are not to be construed as limiting the application.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present application, unless otherwise indicated, the meaning of "a plurality" is two or more.
In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present application, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
Detailed description of the use of the olfactory receptor of the application in the recognition of trimethylamine and method for detecting trimethylamine
The application provides an application of an olfactory receptor in recognizing trimethylamine, an application of trimethylamine in activating the olfactory receptor, a method for detecting trimethylamine, a method for detecting freshness of meat products, a detection device, an electronic device and an executable storage medium, which are respectively described in detail below.
Use of the same
In a first aspect of the application, the application proposes the use of the olfactory receptor rTAAR8c for the recognition of trimethylamine. The application adopts trimethylamine to stimulate the olfactory receptor rTAAR8c, and then can effectively activate rTAAR8c, so that the application adopts the olfactory receptor rTAAR8c to identify the trimethylamine, can be used for detecting whether the sample contains the trimethylamine, and lays a foundation for the identification of the trimethylamine and the detection of the sample containing the trimethylamine.
It should be noted that, if the olfactory receptor is activated after the trimethylamine stimulates the olfactory receptor, the olfactory receptor is "the olfactory receptor can recognize trimethylamine"; if the olfactory receptor is not activated after the trimethylamine stimulates the olfactory receptor, the olfactory receptor is "the olfactory receptor can not recognize the trimethylamine".
According to an embodiment of the present application, the above-mentioned use may further include at least one of the following technical features:
according to an embodiment of the application, the rTAAR8c amino acid sequence has GenBank ID number Q923Y0.1.
According to an embodiment of the application, said recognition is manifested by a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
According to an embodiment of the application, the recognition is manifested by an increase in calcium ions of the cells. Experiments show that when the trimethylamine stimulates cells expressing the olfactory receptor, the concentration of calcium ions (Ca 2+) in the cells is increased after the olfactory receptor is activated, and whether the olfactory receptor can recognize the trimethylamine can be determined by detecting the change of the concentration of the calcium ions.
In some alternative embodiments of the application, the recognition may also be manifested by an increase in cAMP downstream of the olfactory receptor.
In a second aspect of the application, the application proposes the use of trimethylamine for activating the olfactory receptor rTAAR8 c. The application adopts trimethylamine to stimulate the olfactory receptor rTAAR8c, and then can effectively activate rTAAR8c, so that the application adopts the olfactory receptor rTAAR8c to identify the trimethylamine, can be used for detecting whether the sample contains the trimethylamine, and lays a foundation for the identification of the trimethylamine and the detection of the sample containing the trimethylamine.
According to an embodiment of the present application, the above-mentioned use may further include at least one of the following technical features:
According to an embodiment of the application, the activation is manifested by a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
According to an embodiment of the application, the activation is manifested by an increase in calcium ions of the cells. Experiments show that after the trimethylamine stimulates cells expressing an olfactory receptor, the concentration of calcium ions (Ca 2+) in the cells is increased after the olfactory receptor is activated, and whether the trimethylamine can activate the olfactory receptor rTAAR8c can be determined by detecting the change of the concentration of the calcium ions.
In some alternative embodiments of the application, the activation may also be manifested by an increase in cAMP downstream of the olfactory receptor.
Method of
In a third aspect of the application, the application provides a method for detecting trimethylamine. According to an embodiment of the application, the method comprises: contacting a sample to be tested with an olfactory receptor rTAAR8c, and determining a response value of the olfactory receptor rTAAR8c after contact; and determining whether trimethylamine is contained in the sample to be detected based on the response value. From the foregoing, it can be seen that trimethylamine activates the olfactory receptor rTAAR8c, and thus, the sample to be tested is contacted with the olfactory receptor rTAAR8c, if trimethylamine is contained in the sample to be tested, the olfactory receptor rTAAR8c can be activated, and a response value of the olfactory receptor rTAAR8c after activation is obtained, and whether trimethylamine is contained in the sample to be tested can be determined according to the response value. The detection method has the advantages of high response strength, high sensitivity, high accuracy and the like.
According to an embodiment of the present application, the above method may further include at least one of the following technical features:
it should be noted that "contact" in this specification is to be understood in a broad sense, and may be a direct contact or an indirect contact, and is not particularly limited.
According to the embodiment of the application, the sample to be measured can be liquid or gas, and the specific type is not limited, and the sample to be measured is within the protection scope of the application.
Illustratively, the sample to be tested (either liquid per se or prepared as a liquid using a solvent) is contacted with the olfactory receptor after being mixed in a liquid state (i.e., the sample to be tested is in direct contact with the olfactory receptor); or directly placing the sample to be tested and the olfactory receptor in the same space, wherein the odor molecules (trimethylamine) released by the sample to be tested are contacted with the olfactory receptor (namely, the sample to be tested and the olfactory receptor are indirectly contacted); it is also possible to place the olfactory receptor in a space in which a certain amount of gas is released. In short, the type of the sample to be measured is not particularly limited, and the contact mode of the sample to be measured and the olfactory receptor is not particularly limited, and the sample to be measured and the olfactory receptor are all within the protection scope of the application.
According to an embodiment of the present application, the sample to be tested includes, but is not limited to, food or an additive thereof, medicine or a raw material thereof, an auxiliary material or an intermediate thereof, a pesticide or a raw material thereof, a disinfectant, an alarm agent for natural gas, an analytical reagent, a photographic material, a rubber aid, an explosive, a chemical fiber solvent, a surfactant, a dye or a raw material thereof, and the like.
According to an embodiment of the present application, the presence response value of the olfactory receptor rTAAR8c is an indication that trimethylamine is contained in the sample to be tested, or the absence response value of the olfactory receptor rTAAR8c is an indication that trimethylamine is not contained in the sample to be tested.
It should be noted that the "indication without trimethylamine" means that trimethylamine is not present in the sample to be measured; or a small amount of trimethylamine is present in the sample to be measured but cannot be detected.
Illustratively, the olfactory receptor is rTAAR8c and the absence of a response value for the olfactory receptor is a concentration of trimethylamine in the sample to be tested of less than 10. Mu.M.
According to an embodiment of the application, the method further comprises: and determining the content of trimethylamine in the sample to be detected based on a standard curve, wherein the standard curve is a corresponding curve of a preset amount of trimethylamine and an olfactory receptor rTAAR8c response value.
According to an embodiment of the application, the olfactory receptor rTAAR8c is provided by a cell or transgenic cell expressing the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
In some alternative embodiments of the application, the eukaryotic cells include, but are not limited to, cells selected from the group of cells isolated from the olfactory substrate, HEK293 cells, CHO cells, xenopus oocytes, hela cells, COS cells, yeast cells, and the like.
In some alternative embodiments of the application, the prokaryotic cell is selected from bacteria.
According to an embodiment of the application, the response value is obtained by detecting a change in the concentration of Ca 2+ in the cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of olfactory receptor protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
According to an embodiment of the application, the change in activity of the olfactory receptor is obtained by a change in calcium ions of the cell. Experiments show that when the trimethylamine stimulates cells expressing the olfactory receptor, the concentration of calcium ions in the cells is increased after the olfactory receptor is activated, and whether the olfactory receptor can detect the trimethylamine can be determined by detecting the concentration change of the calcium ions.
For example, the Ca 2+ concentration assay is performed by stimulating cells containing an olfactory receptor with trimethylamine or a sample containing trimethylamine, and if the olfactory receptor is activated, the intracellular Ca 2+ level is increased, so that the response of the olfactory receptor can be characterized by a calcium ion flow assay to determine whether the sample to be tested contains trimethylamine.
In a fourth aspect of the present application, a method of detecting freshness of a meat product is provided. According to an embodiment of the application, the method comprises: contacting meat food to be detected with an olfactory receptor rTAAR8c, and determining a response value of the olfactory receptor rTAAR8c after contact; and determining the freshness of the meat food to be tested based on the response value. From the foregoing, it can be seen that trimethylamine can activate the olfactory receptor rTAAR8c, and thus, the sample to be tested is contacted with the olfactory receptor rTAAR8c, if trimethylamine is contained in the sample to be tested, the olfactory receptor rTAAR8c can be activated, a response value of the olfactory receptor rTAAR8c after activation is obtained, trimethylamine is contained in the food to be tested can be determined based on the response value, and the freshness of the meat food to be tested can be judged according to the trimethylamine. The method has the advantages of simple operation, rapid detection, high response strength, strong sensitivity, high accuracy and the like.
According to an embodiment of the present application, the above method may further include at least one of the following technical features:
According to an embodiment of the application, the meat food to be tested includes, but is not limited to, seafood, beef, mutton, pork.
According to an embodiment of the application, the olfactory receptor rTAAR8c presence response value is an indication that the meat product under test is not fresh, or the olfactory receptor rTAAR8c absence response value is an indication that the meat product under test is fresh.
According to an embodiment of the application, the method further comprises: and determining the content of trimethylamine in the meat food to be detected based on a standard curve, wherein the standard curve is a curve corresponding to the response value of the predetermined amount of trimethylamine and the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the olfactory receptor rTAAR8c is provided by a cell or transgenic cell expressing the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
In some alternative embodiments of the application, the eukaryotic cells include, but are not limited to, cells selected from the group of cells isolated from the olfactory substrate, HEK293 cells, CHO cells, xenopus oocytes, hela cells, COS cells, yeast cells, and the like.
In some alternative embodiments of the application, the prokaryotic cell is selected from bacteria.
According to an embodiment of the application, the response value is obtained by detecting a change in the concentration of Ca 2+ in the cell.
According to an embodiment of the application, the response value is obtained by detecting a change in the activity of the olfactory receptor rTAAR8 c.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is obtained based on a change in concentration or value of at least one of the olfactory receptor rTAAR8c protein conformation, binding or dissociation of G protein, cAMP, IP3, calcium ion, current, pH.
According to an embodiment of the application, the change in activity of the olfactory receptor is obtained by a change in calcium ions of the cell. Experiments show that trimethylamine stimulates cells expressing an olfactory receptor rTAAR8c, the concentration of calcium ions in the cells is increased after the olfactory receptor rTAAR8c is activated, and the freshness of meat food to be detected can be determined by detecting the change of the concentration of the calcium ions.
According to an embodiment of the application, the change in activity of the olfactory receptor rTAAR8c is determined by at least one of the following detection methods: luciferase assay, secreted alkaline phosphatase assay, fluorescent protein assay, fluorescent probe assay, ca 2+ concentration assay, amperometric assay, isotopic labeling method, antibody assay and pH assay.
Detection device, electronic apparatus, executable storage medium
In a fifth aspect of the application, the application proposes a detection device for detecting freshness of meat products and/or for detecting trimethylamine. According to an embodiment of the present application, the detection device includes: a detection unit for acquiring a response value of an olfactory receptor rTAAR8c to a sample to be tested, wherein the detection unit contains the olfactory receptor rTAAR8c; and the determining unit is used for determining whether trimethylamine is contained in the sample to be detected or whether the sample to be detected is fresh or not based on the response value. Therefore, whether the sample to be detected contains trimethylamine or not can be detected rapidly and accurately, or whether the sample to be detected (such as meat sample) is fresh or not can be detected rapidly and accurately.
In a sixth aspect of the application, the application provides an electronic device. According to an embodiment of the present application, the electronic device includes: a memory and a processor; the memory is used for storing a computer program; the processor is configured to execute the computer program to implement the method according to the third or fourth aspect. The electronic equipment can be used for detecting whether the sample to be detected contains trimethylamine or not or determining whether the sample to be detected (such as a meat sample) is fresh or not, and has the advantages of simplicity and convenience in operation, rapidness in identification, high accuracy and the like.
According to an embodiment of the application, the electronic device may be an electronic nose device.
In a seventh aspect of the present application, the present application proposes an executable storage medium. According to an embodiment of the application, the storage medium stores computer program instructions which, when run on a processor, cause the processor to perform the method according to the third or fourth aspect.
The scheme of the present application will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present application and should not be construed as limiting the scope of the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: screening of olfactory receptors
In biological organisms, calcium ions produce a wide variety of intracellular signals that are present in almost every type of cell and play an important role in many functions. The principle of the calcium imaging technology is that the calcium ion concentration in the cells can be expressed by fluorescence intensity by utilizing the fluorescent probe by means of the corresponding relation between the calcium ion concentration and the cell activity, so that the purpose of monitoring the cell activity is achieved.
The calcium ion flow detection method (abbreviated as calcium flow method) is a method for detecting the intracellular Ca 2+ level in real time by using a high-sensitivity fluorescent probe. When the olfactory receptor on the cell membrane is activated, the intracellular Ca 2+ level is increased, so that the activity of the olfactory receptor can be related to the Ca 2+ level, and the activated state of the olfactory receptor can be detected in real time by using a calcium ion flow detection method. The calcium ion flow detection method is used for combining cells or cell extracts expressing the olfactory receptor in response to trimethylamine, and a product or method for rapidly detecting trimethylamine is built.
In this example, gene constructs of animal (human, mouse, rat or zebra fish) olfactory receptor, G protein, helper protein bRTP S were transfected into HEK293T cells with transfection reagent lipo2000, after 24 hours, cells were incubated with fluorescent probes (MD-R8190), stimulated with trimethylamine at different concentrations and detected in real time.
TAAR is a trace amine olfactory receptor, and a pool of TAAR olfactory receptors of four species, human, mouse, rat and zebra fish, was stimulated with trimethylamine diluted to a concentration of 1000. Mu.M with HBSS buffer (Gibco-14025076), and screening experiments were performed using calcium flux to finally obtain 3 responding olfactory receptors, i.e., hTAAR5, mTAAR5 and rTAAR8c, see FIG. 1 in particular.
Example 2: detection of activation of olfactory receptors by trimethylamine at different concentrations
1. This example uses calcium flow to determine the response of trimethylamine concentrations (e.g., 0.01mM, 0.03mM, 0.10 mM, 0.30 mM, 1.00 mM) to the olfactory receptor hTAAR5 (amino acid sequence, genBank ID No. NP-001376456.1), mTAAR5 (amino acid sequence, genBank ID No. NP-001009574.1) and rTAAR8c, respectively, and measures the response of these three olfactory receptors at varying concentrations of trimethylamine, see example 1, for a specific test result, see FIG. 2 (where the horizontal axis is the gradient of trimethylamine concentration (mM) and the vertical axis is the fold change in response relative to the blank (no trimethylamine stimulation)). FIG. 2 shows a dose-dependent bar graph of hTAAR5, mTAAR5 and rTAAR8c responses to trimethylamine; the results show that the maximal response fold change of hTAAR5, mTAAR5 and rTAAR8c to trimethylamine can be up to about 2.5 fold, and for sensitivity, the reaction can be initiated at 0.01 mM. Also, the olfactory receptor rTAAR8c has a stronger response intensity than the olfactory receptors hTAAR5 and mTAAR 5.
2. Furthermore, the inventors performed a lateral comparison of the olfactory receptors hTAAR5, mTAAR5 and rTAAR8c, i.e. the detection of calcium ion flux signals at 1mM concentration of trimethylamine stimulation using cells expressing the olfactory receptors hTAAR5, mTAAR5 or rTAAR8c, respectively, wherein the specific detection method is described in example 1. And cells that did not express any olfactory receptor were used as a control, and the results of the detection are shown in FIG. 3. From the results of fig. 3, it can be seen that over time, the cells produced a pronounced calcium flux signal and that the three olfactory receptors exhibited a strong or weak difference in response, especially a significant increase in the multiple of the rTAAR8c response.
Thus, it can be further demonstrated that trimethylamine can be rapidly, sensitively and specifically detected using a cell-binding calcium ion stream assay that expresses the olfactory receptor, rTAAR8 c.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.
Claims (6)
1. A method for detecting trimethylamine, comprising:
Contacting a sample to be tested with an olfactory receptor, rTAAR8c, and determining a response value of said olfactory receptor, rTAAR8c, after contacting, wherein the amino acid sequence of said rTAAR8c has GenBank ID number Q923Y0.1, said olfactory receptor, rTAAR8c, being provided by a cell or transgenic cell expressing said olfactory receptor, rTAAR8c, said response value being obtained by detecting a change in the concentration of Ca 2+ in said cell;
determining whether trimethylamine is contained in the sample to be detected based on the response value;
The presence response value of the olfactory receptor rTAAR8c is an indication that the sample to be tested contains trimethylamine, or the absence response value of the olfactory receptor rTAAR8c is an indication that the sample to be tested does not contain trimethylamine.
2. The method according to claim 1, wherein the method further comprises:
And determining the content of trimethylamine in the sample to be detected based on a standard curve, wherein the standard curve is a corresponding curve of a preset amount of trimethylamine and an olfactory receptor rTAAR8c response value.
3. The method of claim 1, wherein the cell or transgenic cell is a eukaryotic cell or a prokaryotic cell.
4. The method of claim 1, wherein the change in activity of the olfactory receptor rTAAR8c is determined by a Ca 2+ concentration assay.
5. An electronic device, comprising: a memory and a processor;
The memory is used for storing a computer program;
The processor is configured to execute the computer program to implement the method according to any one of claims 1 to 4.
6. An executable storage medium having stored thereon computer program instructions which, when run on a processor, cause the processor to perform the method of any of claims 1 to 4.
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