WO2023162859A1 - Procédé d'inspection et dispositif d'inspection - Google Patents

Procédé d'inspection et dispositif d'inspection Download PDF

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Publication number
WO2023162859A1
WO2023162859A1 PCT/JP2023/005542 JP2023005542W WO2023162859A1 WO 2023162859 A1 WO2023162859 A1 WO 2023162859A1 JP 2023005542 W JP2023005542 W JP 2023005542W WO 2023162859 A1 WO2023162859 A1 WO 2023162859A1
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WO
WIPO (PCT)
Prior art keywords
light
marker
specific antibody
saliva
attached
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PCT/JP2023/005542
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English (en)
Japanese (ja)
Inventor
吾 根武谷
健 浦野
義明 岩下
Original Assignee
国立大学法人島根大学
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Application filed by 国立大学法人島根大学 filed Critical 国立大学法人島根大学
Publication of WO2023162859A1 publication Critical patent/WO2023162859A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to an inspection method and an inspection apparatus.
  • the testing methods that are actually used take a long time, about 5 hours for PCR testing and about 15 minutes for antigen testing, and the testing cost is high. Therefore, a more suitable method for detecting viruses in an unspecified number of subjects has been desired.
  • the present invention has been made in view of these points, and aims to enable rapid testing at low cost to determine whether a subject is infected with a virus.
  • the specific antibody with a marker is an antibody for binding to the antigen with the spike protein of the virus
  • a fluorophore that emits fluorescence upon irradiation is bound, and the dye-marker-attached specific antibody reflects or absorbs the antibody for binding to the spike protein-attached antigen of the virus and the first light.
  • the reagent comprises a specific antibody with a first dye marker to which the antibody and a first dye of a first color are bound, and a second dye of a second color different from the antibody and the first color. and a second dye-marked specific antibody to which is bound, and in the determining step, in the second light generated from one of the conjugates, the first color light and the second It may be determined that the virus is contained in the saliva of the subject according to the presence of light of a color of .
  • an image of the second light is captured, and in the determination step, the second It may be determined that the virus is contained in the saliva of the subject according to the inclusion of pixels of one color and pixels of the second color.
  • the step of spraying a buffer solution or water onto the mixed solution of the saliva and the reagent is further provided, and in the step of irradiating the first light, the buffer solution or water is sprayed onto the mixed solution of the first of light may be applied.
  • the step of applying a magnetic field to at least a part of the mixed liquid so that the conjugate moves to a predetermined region In the step of detecting the intensity of the virus, the intensity of the second light generated from the predetermined region by the irradiation of the first light is detected, and the reagent is the marker-attached specific antibody, It may further comprise a magnetic marker-attached specific antibody in which the antibody for binding to the spike protein-attached antigen and a magnetic substance are bound.
  • a groove of a predetermined shape is provided in the region where the saliva and the reagent are mixed on the one surface of the test rod.
  • the magnetic field may be applied to move the conjugate along a direction to move the conjugate to the predetermined region in the groove.
  • the intensity of the second light is increased while moving the combined body in the step of applying the magnetic field.
  • the detecting step may detect the intensity of the second light received from the conjugate moving in the predetermined area.
  • the saliva adhered to the test rod by the subject may be sprayed with the reagent containing the specific antibody with the marker.
  • the reagent containing the marker-attached specific antibody may be dropped onto the saliva adhered to the test rod by the subject.
  • the reagent corresponding to the type of the marker-attached specific antibody may be sprayed or dropped among a plurality of the reagents different for each type of the marker-attached specific antibody.
  • the mixing step may include a step in which the subject attaches the saliva of the subject to the one surface of the test rod to which the reagent containing the marker-attached specific antibody is previously attached.
  • a step of vibrating the test rod may be further included after the mixing step.
  • the test rod has a material that is transparent to the first light and the fluorescence, and in the step of irradiating the first light, the test rod is attached to the surface of the test rod with the saliva attached to the subject. may be irradiated with the first light from the opposite surface.
  • an insertion opening for a subject to insert a test rod having saliva attached to one surface thereof, and an area of the test rod inserted into the insertion opening to which the saliva is attached an attachment unit for attaching a reagent containing a specific antibody with a marker and mixing the saliva and the reagent; a light source unit for irradiating a mixture of the saliva and the reagent with a first light; and the marker A detection unit that detects the second light generated by irradiating the first light to the conjugate in which the specific antibody has multiple bonds with the virus having the antigen with the spike protein, and based on the detected light intensity, a determination unit that determines whether the virus is contained in the saliva of the subject, and the reagent is at least one of a specific antibody with a fluorescent marker and a specific antibody with a dye marker as the specific antibody with a marker.
  • the specific antibody with a fluorescent marker binds an antibody for binding to the spike protein-attached antigen of the virus and a fluorophore that emits fluorescence when irradiated with the first light.
  • the dye-marker-attached specific antibody includes the antibody for binding to the spike protein-attached antigen of the virus and a dye that reflects or absorbs the first light.
  • the reagent comprises a specific antibody with a first dye marker to which the antibody and a first dye of a first color are bound, and a second dye of a second color different from the antibody and the first color. and a second dye-marker-attached specific antibody to which is bound, the determination unit includes, in the second light generated from one of the conjugates, the light of the first color and the second light of the It may be determined that the virus is contained in the saliva of the subject according to the presence of colored light.
  • the detection unit has an imaging unit that captures the second light, and the determination unit selects pixels of the imaging region in which the combined body is captured from the captured image captured by the imaging unit. It may be determined that the virus is contained in the saliva of the subject according to the inclusion of pixels of one color and pixels of the second color.
  • a sprayer for spraying a buffer solution or water onto the mixture of the saliva and the reagent may be further provided.
  • a magnetic field application unit that applies a magnetic field to at least a part of the mixed liquid so that the combined body moves to a predetermined area, and the detection unit detects the predetermined area by irradiating the first light.
  • the intensity of the second light generated from the region is detected, and the reagent is the marker-attached specific antibody, in which the antibody for binding to the spike protein-attached antigen of the virus and a magnetic substance are bound. It may further include a magnetic marker-attached specific antibody.
  • a groove having a predetermined shape is provided in a region where the test subject attaches the saliva to the test rod, and the magnetic field applying section extends along the longitudinal direction of the groove of the test rod.
  • the magnetic field may be applied to move the conjugate to the predetermined region in the groove.
  • the width of the groove provided in the test rod may become narrower toward the end in the longitudinal direction of the groove.
  • the end of the groove provided in the test bar may have a V-shape or U-shape protruding closer to the end in the longitudinal direction of the groove in plan view.
  • the attachment section may have a sprayer that sprays the reagent containing the marker-attached specific antibody onto the saliva adhered to the test rod by the subject.
  • the attachment section has a plurality of sprayers for spraying the reagent corresponding to the type of the specific antibody with the marker, and the plurality of sprayers spray the reagent onto the saliva attached to the test rod. good too.
  • the attaching part may have a dropping part for dropping the reagent containing the specific antibody with the marker onto the saliva attached to the test rod.
  • the attaching section moves the attaching section to a position corresponding to each of the insertion openings by the moving section, and then the reagent is dispensed by the attaching section.
  • control unit that detects the intensity of light, wherein the determination unit detects the presence of the virus in the plurality of subjects based on the detection results of the detection unit moved to a plurality of positions corresponding to the plurality of insertion openings.
  • It may further include a vibrating section that vibrates the test rod to mix the saliva and the reagent.
  • the test rod has a material transparent to the first light and the second light, and the light source part is opposite to the surface of the test rod on which the subject adheres the saliva.
  • the first light may be applied from the side surface.
  • FIG. 1 shows a configuration example of an inspection apparatus 100 according to this embodiment.
  • An example of a mixed liquid in which saliva of a subject 10 and a reagent containing a specific antibody 40 with a marker are mixed is shown.
  • 1 shows a configuration example of a test rod 20 according to this embodiment.
  • An example of the operation flow of the inspection apparatus 100 according to the present embodiment is shown.
  • a modification of the test rod 20 according to the present embodiment is shown.
  • a modification of the operation flow of the inspection apparatus 100 according to the present embodiment is shown.
  • An example of the distribution of the number of particles with respect to the particle size of particles detected by the inspection apparatus 100 according to the present embodiment is shown.
  • An example of normalizing the distribution of the number of particles with respect to the particle size of particles detected by the inspection apparatus 100 according to the present embodiment is shown.
  • An example in which a virus 30 binds to a marker-attached specific antibody 40 according to this embodiment is shown.
  • a first example for comparison is shown.
  • a second example for comparison is shown.
  • a third example for comparison is shown.
  • An optical microscope image shows an example in which a combined body 50 according to the present embodiment is formed.
  • FIG. 1 shows an outline of an examination method for a subject by an examination apparatus 100 according to this embodiment.
  • the testing device 100 tests whether or not the subject 10 is infected with a virus having an antigen with a spike protein, such as the novel coronavirus.
  • the subject 10 adheres saliva of the subject 10 to a predetermined position on one surface of the test rod 20 ((1) in FIG. 1).
  • the test subject 10 for example, licks the test stick 20 so that his/her own saliva adheres to it.
  • the subject 10 or a person other than the subject 10 may put the test rod 20 into the mouth of the subject 10 to allow saliva of the subject 10 to adhere to the test rod 20 .
  • the subject 10 inserts the test rod 20 with saliva attached into the insertion port 110 of the inspection device 100 ((2) in FIG. 1).
  • the inspection device 100 inspects whether or not the saliva adhering to the test rod 20 inserted into the insertion port 110 contains a virus having a spike protein-attached antigen ((3) in FIG. 1).
  • a plurality of insertion openings 110 may be provided in the inspection device 100 .
  • a plurality of different subjects 10 may insert test rods 20 to which their own saliva is attached into a plurality of insertion openings 110 respectively.
  • the inspection device 100 inspects whether the saliva adhering to the plurality of test rods 20 inserted into the plurality of insertion openings 110 contains viruses.
  • the display unit 190 of the inspection device 100 displays the inspection results of the inspection device 100 ((4) in FIG. 1). As described above, according to the testing device 100 according to the present embodiment, by inserting the test rod 20 to which the saliva of the subject 10 is attached into the insertion port 110 of the testing device 100, the subject 10 is infected with the virus. You can check whether there is Such an inspection apparatus 100 will be described below.
  • FIG. 2 shows a configuration example of an inspection apparatus 100 according to this embodiment.
  • the inspection apparatus 100 irradiates light of a predetermined wavelength while applying a magnetic field to the mixture of the saliva and the reagent, thereby detecting viruses in the saliva.
  • the inspection apparatus 100 includes an insertion port 110, an attachment section 120, a vibration section 130, a light source section 140, a magnetic field application section 150, a detection section 160, a storage section 170, a control section 180, and a display section 190. Prepare.
  • the test rod 20 to which the subject 10 has attached the saliva of the subject 10 is inserted into the insertion port 110 .
  • a guide or the like for stabilizing the position of the inserted test rod 20 is desirably provided inside the insertion port 110 or the inspection device 100 .
  • the insertion port 110 and/or the test rod 20 should be provided with a guide or the like so that the test rod 20 is inserted into the insertion port 110 with the saliva adhering surface of the test rod 20 facing a predetermined direction. is desirable.
  • the cross section of the insertion port 110 and/or the test rod 20 may be processed so that the test rod 20 cannot be inserted unless the attachment surface is oriented in a predetermined direction.
  • the adhering part 120 adheres a reagent containing a marker-attached specific antibody to the saliva-adhered region of the test rod 20 inserted into the insertion port 110, and mixes the saliva with the reagent.
  • the reagent contains multiple types of specific antibodies with markers. Such a marker-attached specific antibody will be described with reference to FIG.
  • FIG. 3 shows an example of a liquid mixture in which the saliva of the subject 10 and a reagent containing the marker-attached specific antibody 40 are mixed.
  • FIG. 3 shows an example in which virus 30 is contained in saliva of subject 10 .
  • Virus 30 has antigens 31 with multiple spike proteins.
  • Virus 30 is, for example, a novel coronavirus. Such a virus 30 has a diameter of about 10 nm, which is not a size that can be quickly confirmed by direct observation with a microscope or the like.
  • the marker-equipped specific antibody 40 also has a diameter of about several tens of nanometers, which is not a size that can be quickly confirmed by direct observation with a microscope or the like.
  • a specific antibody 40 with a marker has a plurality of antibodies 41 and markers 42 .
  • a plurality of antibodies 41 are arranged around the marker-attached specific antibody 40 , and the plurality of antibodies 41 and the markers 42 are bound together so that the markers 42 are positioned inside.
  • Antibody 41 binds to spike protein-attached antigen 31 of virus 30 .
  • the adhering portion 120 mixes the saliva of the subject 10 with the reagent and the virus 30 and the specific antibody 40 with a marker come into contact with each other, the antigen 31 with the spike protein of the virus 30 binds to the antibody 41 of the specific antibody 40 with the marker. .
  • a conjugate 50 of the virus 30 and the marker-attached specific antibody 40 is formed.
  • Viruses 30 have a plurality of spike protein-attached antigens 31, and marker-attached specific antibodies 40 have a plurality of antibodies 41. Therefore, when a plurality of viruses 30 are present in a mixture, a plurality of viruses 30 and a plurality of marker-attached specific antibodies 40 A conjugate 50 is formed in which is multiple-bonded. The diameter of such multiple-bonded conjugates can be on the order of several hundred nanometers. Also, a plurality of such conjugates 50 are formed in the liquid mixture. Since the antibodies 41 do not bind to each other, the combined body 50 that is large enough for direct observation is not formed unless a plurality of viruses 30 are present in the mixture.
  • the marker 42 is an index used to facilitate detection of such a conjugate 50.
  • the marker 42 is, for example, a magnetic material, a fluorescent material, a dye, or the like.
  • the marker-attached specific antibody 40 using a magnetic material as the marker 42 is called a magnetic marker-attached specific antibody.
  • the magnetic marker-attached specific antibody is the marker-attached specific antibody 40 in which an antibody for binding to the spike protein-attached antigen 31 of the virus 30 and a magnetic substance are bound.
  • a magnetic material is a material that, when a magnetic field is applied from the outside, is magnetized in response to the applied magnetic field.
  • a specific antibody 40 with a marker that uses a fluorescent substance as the marker 42 is called a specific antibody with a fluorescent marker.
  • the fluorescent marker-attached specific antibody is the marker-attached specific antibody 40 in which an antibody for binding to the spike protein-attached antigen 31 of the virus 30 and a fluorescent substance are bound.
  • a phosphor is a substance that emits fluorescence with a wavelength longer than a predetermined wavelength when irradiated with light with a wavelength shorter than the predetermined wavelength.
  • light having a wavelength shorter than a predetermined wavelength is used as the first light
  • fluorescence is used as the second light.
  • the first light is light having energy greater than a predetermined energy and functions as excitation light in this case.
  • the specific antibody 40 with a marker that uses a dye as the marker 42 is called a specific antibody with a dye marker.
  • the specific antibody with a pigment marker is the specific antibody with a marker 40 in which an antibody for binding to the antigen 31 with spike protein of the virus 30 and a pigment that reflects or absorbs the first light are bound.
  • reflected light obtained by the dye reflecting the first light or passing light obtained by the first light passing through the dye is used as the second light.
  • the reagent contains a magnetic marker-attached specific antibody as the marker-attached specific antibody 40 .
  • the reagent further includes at least one of a specific antibody with a fluorescent marker and a specific antibody with a dye marker as a specific antibody with a marker.
  • the above-described marker-attached specific antibody 40 was discovered by the applicants. In this embodiment, first, an example in which the marker-attached specific antibody 40 includes a magnetic marker-attached specific antibody and a fluorescent marker-attached specific antibody will be described.
  • the adhering part 120 mixes the above-described reagent with saliva on one side of the test rod 20 .
  • the attachment section 120 has, for example, a sprayer that sprays a reagent containing the marker-attached specific antibody 40 onto the saliva attached to the test rod 20 by the subject 10 .
  • the applicator 120 may have multiple atomizers.
  • the atomizer atomizes the reagent and sprays it onto the area of the test rod 20 where saliva is attached.
  • the sprayer is, for example, a head part of an ink-jet printer, a spray nozzle, or the like, which sprays a reagent by the piezoelectric effect of piezo, heating, pressure by a pump, or the like.
  • Such a sprayer can efficiently adhere the reagent to the area where the saliva adheres and mix the reagent with the saliva while reducing the amount of the reagent used.
  • the vibrating section 130 vibrates the test rod 20 to which the reagent is attached by the attaching section 120 to mix the saliva and the reagent.
  • the vibrating section 130 vibrates the test rod 20 so as to agitate the saliva and the reagent. Thereby, the vibrating section 130 can reduce uneven mixing of the saliva and the reagent.
  • the inspection device 100 may include a heating section that heats the test rod 20 .
  • Heating causes molecules contained in the saliva and the reagent to vibrate (Brownian motion), making it easier for the virus 30 and the marker-attached specific antibody 40 to bind.
  • the heating unit heats the molecules contained in the saliva and the reagent to cause molecular motion, and shortens the distance between the antigen 31 with the spike protein of the virus 30 and the antibody 41 of the specific antibody 40 with the marker. The binding probability between the virus 30 and the marker-attached specific antibody 40 can be improved.
  • the light source unit 140 irradiates the first light to at least part of the region of the test rod 20 to which saliva is attached, and irradiates the mixture of saliva and the reagent with the first light.
  • the light source unit 140 has a laser, an LED, or the like.
  • the light source unit 140 outputs, as the first light, light that functions as excitation light for the fluorescent substance contained in the specific antibody with a fluorescent marker, for example.
  • the light source unit 140 may output, as the first light, light reflected or absorbed by the pigment contained in the specific antibody with a pigment marker.
  • the light source unit 140 outputs, for example, ultraviolet light as the first light.
  • the light source unit 140 may output visible light as the first light. Also, the light source unit 140 may output light of a plurality of wavelengths as the first light.
  • the fluorescent substance contained in the specific antibody with the fluorescent marker By the irradiation of the first light from the light source unit 140, the fluorescent substance contained in the specific antibody with the fluorescent marker generates fluorescence (second light). Since the diameter of the phosphor is as small as several tens of nanometers or less, the light intensity of the generated second light is also small. On the other hand, since the multiply-bonded conjugate 50 contains a plurality of specific antibodies with fluorescent markers, the light intensity of the second light generated from the conjugate 50 is several tens to several hundred times more. can be.
  • the magnetic field application unit 150 applies a magnetic field to the mixture of saliva and reagent.
  • the magnetic field applying section 150 applies a magnetic field of a predetermined magnitude to a region of the test rod 20 to which the light source section 140 is irradiating the first light.
  • the magnetic field applying unit 150 applies a magnetic field parallel to the first direction, as indicated by arrows in FIG.
  • the magnetic field applying section 150 has, for example, a plurality of coils arranged to sandwich the test rod 20 . Then, the magnetic field applying unit 150 applies a magnetic field to the liquid mixture by causing currents to flow through the plurality of coils.
  • the magnetic field applying section 150 may have a movable permanent magnet and apply the magnetic field to the liquid mixture by bringing the permanent magnet close to the test rod 20 .
  • the specific antibody with a magnetic marker is contained in the specific antibody with a magnetic marker 40. Therefore, when a magnetic field in the first direction is applied to the mixed solution, the specific antibody with a magnetic marker in the mixed solution will move in the first direction. A directional force is applied. Since the marker-attached specific antibody 40 floats in the mixture, it moves in the first direction. In addition, since the multiply-bonded conjugate 50 contains a plurality of specific antibodies with magnetic markers, it moves in the first direction. Such movement of the magnetic marker-attached specific antibody and the conjugate 50 is called magnetophoresis. Since the specific antibody with a fluorescent marker does not contain a magnetic substance, it does not move even when a magnetic field is applied.
  • the magnetic field applying unit 150 applies at least a portion of the mixture so that the conjugate 50, in which the specific antibody 40 with the marker and the virus 30 having the antigen 31 with the spike protein are multiply bound, moves to a predetermined region.
  • the predetermined area is the area on the first direction side among the areas of the test rod 20 to which saliva adheres, as will be described later.
  • the magnetic field applying unit 150 applies a magnetic field, the binder 50 in the liquid mixture moves to a predetermined area.
  • the application of the magnetic field by the magnetic field applying section 150 can collect the plurality of combined bodies 50 in a predetermined region of the liquid mixture.
  • the detection unit 160 detects the intensity of the second light generated from a predetermined area by irradiating the first light. Since a plurality of conjugates 50 are gathered in a predetermined region of the liquid mixture, the intensity of the second light generated by irradiation with the first light is the same as that of the second light when the conjugates 50 are not present. Compared to the intensity of Therefore, the detection unit 160 can receive and easily detect the second light generated from the plurality of conjugates 50 .
  • the detection unit 160 is, for example, a photodiode, CCD, or the like.
  • the optical system such as the light source section 140 and the detection section 160 is desirably fixed to the fixing section 162 .
  • the attaching portion 120 may also be fixed to the fixing portion 162 .
  • the storage unit 170 is a storage medium including ROM (Read Only Memory) and RAM (Random Access Memory).
  • the storage unit 170 may also include a large-capacity storage device such as a HDD (Hard Disk Drive) and/or an SSD (Solid State Drive).
  • a computer functions as the control unit 180, information such as an OS (Operating System) and programs that make the computer function may be stored.
  • the storage unit 170 may store various information including a database that is referred to during program execution.
  • the storage unit 170 may store intermediate data generated (or used) by the inspection apparatus 100 in the process of operation, calculation results, thresholds, reference values, parameters, and the like. In addition, the storage unit 170 may supply the stored data to the request source in response to a request from each unit in the inspection apparatus 100 .
  • the control unit 180 controls each unit of the inspection device 100 .
  • the control unit 180 is, for example, a CPU (Central Processing Unit).
  • the control unit 180 has a determination unit 181 and a display control unit 182 .
  • the CPU functions as the control section 180 having the determination section 181 and the display control section 182 by executing the program stored in the storage section 170 .
  • the determination unit 181 determines whether the virus 30 is contained in the subject's 10 saliva based on the detected light intensity.
  • the conjugate 50 is formed in the mixed liquid, so that, for example, the light intensity detected by the detection unit 160 is as described above. , the light intensity is such that it exceeds the threshold.
  • the determination unit 181 can determine whether or not the saliva of the subject 10 contains the virus 30 by comparing the light intensity level detected by the detection unit 160 with the threshold value.
  • the display control unit 182 controls the display unit 190 to display the determination result of the determination unit 181. For example, when the virus 30 is contained in the subject's 10 saliva, the display control unit 182 causes the display unit 190 to display "positive" or “suspected positive” at the position corresponding to the test rod 20 of the subject 10 . If the virus 30 is not contained in the subject's 10 saliva, the display control unit 182 causes the display unit 190 to display "negative” at the position corresponding to the test rod 20 of the subject 10 .
  • the display unit 190 is a display such as a liquid crystal provided in the inspection device 100 .
  • the display unit 190 may be a display device, monitor device, or the like that is separate from the inspection device 100 .
  • the inspection apparatus 100 may be connected to a network, and the display unit 190 may be provided at a position away from the inspection apparatus 100 .
  • information indicating the determination result may be transmitted to the mobile terminal possessed by the subject, and the determination result may be displayed on the display surface of the mobile terminal.
  • the inspection results of the inspection apparatus 100 may be stored in a database or the like via a network.
  • the inspection device 100 collects the conjugates 50 in which a plurality of viruses 30 and a plurality of marker-attached specific antibodies 40 are multiply bound to a predetermined area by applying a magnetic field.
  • the inspection apparatus 100 can increase the light intensity of weak fluorescence generated from a single marker-attached specific antibody 40 by irradiation with excitation light to several hundred times or more. can be easily determined whether is contained in saliva.
  • the inspection device 100 described above may have a plurality of insertion openings 110, and a plurality of different test rods 20 may be inserted into the insertion openings 110, respectively. In this case, it is desirable that the inspection apparatus 100 can sequentially inspect a plurality of test rods 20 by moving the attachment section 120 , the light source section 140 and the detection section 160 .
  • the inspection apparatus 100 further includes a moving section that sequentially moves the attaching section 120, the light source section 140, and the detecting section 160 to a plurality of positions corresponding to the plurality of insertion openings 110 in one direction.
  • the moving unit for example, moves the attachment unit 120, the light source unit 140, and the detection unit 160 individually. Further, when the light source unit 140 and the detection unit 160 are fixed to the fixing unit 162, the moving unit may move the fixing unit 162 together in one direction.
  • the control unit 180 controls such a moving unit to repeat the operation and movement of the attachment unit 120, the light source unit 140, and the detection unit 160 so as to sequentially inspect a plurality of test rods 20.
  • the magnetic field applying section 150 is arranged so as to be able to apply a magnetic field parallel to the first direction to the plurality of test rods 20 .
  • the inspection device 100 may be provided with a plurality of attachment units 120, light source units 140, and detection units 160.
  • the same number of groups of the attachment section 120 , the light source section 140 , and the detection section 160 as the number of the insertion openings 110 are provided in the inspection apparatus 100 corresponding to the insertion openings 110 .
  • the set of the attachment section 120 , the light source section 140 and the detection section 160 inspects the test rod 20 inserted into the corresponding insertion port 110 .
  • the inspection apparatus 100 can inspect a plurality of test rods 20 in parallel.
  • the inspection apparatus 100 may use the processed test rod 20 to collect the plurality of combined bodies 50 in a narrower area.
  • FIG. 4 shows a configuration example of the test rod 20 according to this embodiment.
  • FIG. 4A shows a front view of the attachment surface 21 on which saliva of the subject 10 is attached.
  • FIG. 4B shows a cross-sectional view along line CC of FIG. 4A.
  • the area where the subject adheres saliva is indicated by a dotted line as an adhesion area 22 .
  • a groove 23 having a predetermined shape is provided in the attachment region 22 .
  • the grooves 23 are desirably provided in the test rod 20 so that the extending direction is parallel to the first direction in which the magnetic field applying section 150 applies the magnetic field.
  • the test rod 20 is inserted into the insertion opening 110 parallel to the longitudinal direction of the test rod 20 .
  • the test rod 20 is arranged in the inspection device 100 such that the first direction in which the magnetic field applying section 150 applies the magnetic field is the X direction in FIG. 4A.
  • the X direction in FIG. 4A is substantially perpendicular to the insertion direction of the test rod 20 and substantially parallel to the attachment surface 21 .
  • the grooves 23 are provided such that their extending directions are substantially perpendicular to the longitudinal direction of the test rod 20 .
  • the magnetic field applying unit 150 applies a magnetic field so that the combined body 50 moves along the longitudinal direction of the groove 23 of the test rod 20, and moves the combined body 50 to a predetermined region in the groove 23.
  • groove 23 has a function of guiding movement of coupling body 50 .
  • the groove 23 provided in the test rod 20 be provided so that the width becomes narrower toward the end in the longitudinal direction of the groove 23 .
  • the groove 23 thereby guides the coupling body 50 to move into a narrower area.
  • the ends of the grooves 23 provided in the test rod 20 may have V-shapes or U-shapes protruding closer to the ends in the longitudinal direction of the grooves 23 in plan view. This allows the grooves 23 to collect the binding bodies 50 in the projecting regions of the longitudinal ends.
  • FIG. 4A shows an example in which the end of the groove 23 has a V shape. Also, in FIG. 4A , a projected region of the end in the longitudinal direction is indicated by oblique lines as a predetermined observation region 24 .
  • the plurality of combined bodies 50 can be collected in such an observation region 24.
  • the light source unit 140 irradiates the observation area 24 with the first light, so that the detection unit 160 can detect the second light with a higher intensity.
  • the test rod 20 preferably has a transparent material with respect to the first light and the second light.
  • at least the observation area 24 of the test rod 20 is made of a transparent material.
  • the light source unit 140 can irradiate the first light from the surface opposite to the adhesion surface 21 of the test rod to which the subject 10 has adhered saliva.
  • the degree of freedom of arrangement of the optical system such as the light source unit 140 and the detection unit 160 can be improved.
  • Such a test rod 20 also makes it possible to detect the transmitted light of the first light in the observation area 24, as will be described later.
  • test rod 20 may have a water retention agent 25 for absorbing saliva. At least part of the water retention agent 25 may be embedded in the grooves 23 . Moreover, the test rod 20 may have a filter 26 for reducing the contamination of the liquid mixture with dust or the like. The filter 26 is provided, for example, so as to overlap the water retention agent 25 .
  • the application of the magnetic field by the magnetic field applying unit 150, the movement of the combined body 50 due to the application of the magnetic field, and the detection of the second light by the detecting unit 160 are operations of several seconds or less. Therefore, the inspection apparatus 100 can output an inspection result for one test rod 20 in several seconds to ten and several seconds, which is several tens to several thousand times longer than the conventional one. An inspection method of such an inspection apparatus 100 will be described below.
  • FIG. 5 shows an example of the operation flow of the inspection apparatus 100 according to this embodiment.
  • the inspection device 100 inspects whether or not the saliva adhering to the test rod 20 contains the virus 30 by executing the operation flow shown in FIG.
  • the operation flow of FIG. 5 is the operation after the plurality of test rods 20 to which the saliva of the subject 10 is attached are inserted into the plurality of insertion openings 110, respectively.
  • the control section 180 controls the moving section to move the attachment section 120 to a predetermined position corresponding to the insertion opening 110 ( S51).
  • the saliva of the subject 10 and a reagent containing the specific antibody 40 with marker are mixed (S52).
  • the marker-attached specific antibody 40 is assumed to include a magnetic marker-attached specific antibody and a fluorescent marker-attached specific antibody.
  • the controller 180 controls the attachment section 120 to spray a reagent containing a specific antibody with a marker onto the saliva attached to the test rod 20 .
  • control section 180 controls the vibration section 130 to vibrate the test rod 20 (S53).
  • the control section 180 controls the vibration section 130 to vibrate the test rod 20 (S53).
  • S52 and S53 By the operations of S52 and S53, when the virus 30 having the spike protein-attached antigen 31 is present in the saliva of the subject 10, multiple binding of the specific antibody 40 with the marker and the virus 30 occurs in the mixture of the saliva and the reagent. Then, a plurality of combined bodies 50 are formed.
  • the conjugate 50 contains a plurality of specific antibodies with magnetic markers, a plurality of specific antibodies with fluorescent markers, and a plurality of viruses 30 .
  • control unit 180 controls the light source unit 140 to irradiate the mixture of saliva and reagent with the first light of a predetermined wavelength (S54). For example, the control unit 180 moves the light source unit 140 and the detection unit 160 to a position corresponding to the first insertion port, and then turns on the light source unit 140 . Note that the control unit 180 may move the light source unit 140 and the detection unit 160 at the stage of S51.
  • the control unit 180 controls the magnetic field applying unit 150 to apply a magnetic field in the first direction to at least part of the mixture of saliva and reagent (S55).
  • the combined body 50 moves to a predetermined area under the force directed in the first direction.
  • the coupling body 50 moves along the longitudinal direction of the groove 23 using the groove 23 of the test rod 20 as a guide.
  • the magnetic field applying unit 150 applies a magnetic field in a first direction parallel to the longitudinal direction of the groove 23, thereby forming a plurality of conjugates 50 in the observation area 24 at the end of the groove 23 in the longitudinal direction, which is a predetermined area. can be collected.
  • control unit 180 controls the detection unit 160 to detect the intensity of the second light received from the predetermined area by the irradiation of the first light (S56).
  • the detection unit 160 detects, as the second light, the fluorescence generated from the phosphor contained in the conjugate 50 collected in the observation region 24 by the irradiation of the first light, which is the excitation light.
  • the determination unit 181 determines whether the virus 30 is contained in the saliva of the subject 10 based on the detected light intensity (S57).
  • the detection unit 160 detects fluorescence intensity exceeding the threshold value, so the determination unit 181 can easily determine the presence or absence of the virus 30.
  • the display control unit 182 causes the display unit 190 to display the determination result (S58).
  • the inspection device 100 repeats the operations from S51 to S58 until the inspection of all the test rods 20 to be inspected inserted into the insertion openings 110 is completed (S59: No).
  • the control unit 180 continues the inspection with the test rod 20 inserted into the insertion opening 110 adjacent to the insertion opening 110 into which the test rod 20 that has been inspected is set as the next inspection object. do.
  • determination unit 181 sequentially determines whether virus 30 is contained in the saliva of multiple subjects 10 based on the detection results of detection unit 160 moved to multiple positions corresponding to multiple insertion openings 110 . judge.
  • the inspection apparatus 100 ends the inspection (S59: Yes).
  • the test rod 20 may be made of a rod-shaped material to which the saliva of the subject 10 can adhere, and can be manufactured at a lower cost than conventional test kits.
  • the test rod 20 may be discarded for infection control.
  • the plurality of insertion openings 110 be integrally formed and provided detachably from the inspection apparatus 100 . Accordingly, the operator of the inspection device 100 can handle the plurality of test rods 20 and the plurality of insertion ports 110 without touching the test rods 20 by removing the integrated insertion port 110 from the inspection device 100. can be done. Therefore, the operator of the inspection device 100 can safely and easily replace the insertion port 110 of the inspection device 100 . Also, the operator can quickly prepare for the next inspection by the inspection apparatus 100 .
  • the reagent contains a magnetic marker-attached specific antibody and a fluorescent marker-attached specific antibody as the marker-attached specific antibody 40
  • the present invention is not limited to this. no.
  • the reagent may include a magnetic marker-attached specific antibody and a dye marker-attached specific antibody as the marker-attached specific antibody 40 .
  • a specific antibody with a dye marker has a dye that reflects the first light. Therefore, when the mixed liquid is irradiated with the first light in S54 and a magnetic field is applied to the mixed liquid in S55 to move the conjugate 50 to the observation region 24, the detection unit 160 detects the reflected light reflected from the dye in S56. is detected as the second light. Accordingly, in S57, the determination unit 181 can determine that the virus 30 is contained in the saliva of the subject 10, for example, when the light intensity detected by the detection unit 160 exceeds the threshold.
  • the dye-marker-attached specific antibody may have a dye that absorbs the first light.
  • the detection unit 160 detects the absorbance of the first light by the liquid mixture.
  • the detection unit 160 detects reflected light reflected from the dye as the second light.
  • the intensity of the second light detected by the detector 160 decreases when the dye absorbs.
  • the determination unit 181 can determine that the virus 30 is contained in the saliva of the subject 10, for example, when the light intensity detected by the detection unit 160 becomes equal to or less than the threshold.
  • the determination unit 181 may use a value obtained by dividing the intensity of the second light by the intensity of the first light that is applied as the absorbance of the first light. In this case, the determination unit 181 determines that the virus 30 is contained in the saliva of the subject 10 when the absorbance becomes equal to or less than the threshold.
  • the detection unit 160 may detect, as the second light, the transmitted light obtained by transmitting the first light through the liquid mixture. For example, when the mixed liquid is irradiated with the first light in S54 and a magnetic field is applied to the mixed liquid in S55 to move the conjugate 50 to the observation region 24, in S56 the detection unit 160 detects the is detected as the second light. In this case, in S57, the determination unit 181 can determine that the virus 30 is contained in the saliva of the subject 10 when the light intensity detected by the detection unit 160 becomes equal to or less than the threshold. Further, the determination unit 181 may calculate the transmittance of the second light and determine that the virus 30 is contained in the saliva of the subject 10 when the transmittance becomes equal to or less than the threshold. .
  • the light source unit 140 may have a plurality of light sources with different wavelengths, and sequentially irradiate the observation area 24 with light from the plurality of light sources as the first light.
  • the detection unit 160 sequentially detects a plurality of reflected light or transmitted light of the first light, and the determination unit 181 calculates a plurality of absorbances corresponding to the plurality of detection results by the detection unit 160 .
  • the determination unit 181 can acquire the absorbance characteristics for each wavelength of the light received from the observation region 24, and determines whether or not the conjugate 50 exists in the observation region 24 according to the absorbance characteristics. I can judge.
  • the determination unit 181 can determine that the virus 30 is contained in the subject's 10 saliva based on the detection result of the detection unit 160 .
  • the test rod 20 preferably has a transparent material with respect to the first light.
  • the light source unit 140 can emit the first light from one surface of the test rod 20 in S54, and the detection unit 160 can detect the second light from the other surface of the test rod 20 in S56.
  • the light source unit 140 irradiates the first light from the surface of the test rod 20 to which the saliva of the subject 10 is attached, opposite to the attachment surface 21 . Thereby, the degree of freedom of arrangement of the light source unit 140 and the detection unit 160 can be improved.
  • the reagent may include a magnetic marker-attached specific antibody, a fluorescent marker-attached specific antibody, and a dye marker-attached specific antibody as the marker-attached specific antibody 40 .
  • the detection unit 160 detects at least one of the fluorescence emitted from the specific antibody with the fluorescent marker, the reflected light emitted from the specific antibody with the dye marker, and the transmitted light from the specific antibody with the dye marker as the second light. detected as the light of The detector 160 may detect two or more types of light, and in this case, the inspection apparatus 100 is provided with a plurality of detectors 160 .
  • the detection unit 160 has a first detection unit that detects fluorescence and a second detection unit that detects reflected light. Then, determination unit 181 determines whether virus 30 is contained in saliva of subject 10 based on the detection result of the first detection unit and the detection result of the second detection unit. For example, when the intensity of the fluorescence detected by the first detection unit exceeds the threshold and the intensity of the reflected light detected by the second detection unit exceeds the threshold, the determination unit 181 detects that the virus 30 is present in the saliva of the subject 10. determined to be included.
  • the detector 160 may have a first detector that detects fluorescence, a second detector that detects reflected light, and a third detector that detects transmitted light.
  • the determination unit 181 can more reliably determine whether the virus 30 is contained in the saliva of the subject 10 based on the intensities of the multiple types of light.
  • the test apparatus 100 has described an example in which a reagent containing a plurality of types of marker-attached specific antibodies 40 is mixed with the saliva of the subject 10, but the present invention is not limited to this.
  • the testing device 100 may mix a plurality of reagents containing different types of marker-attached specific antibodies 40 with the saliva of the subject 10 .
  • the attachment section 120 has a plurality of sprayers that spray reagents corresponding to the types of specific antibodies with markers.
  • the attachment section 120 includes a first sprayer that sprays a reagent containing a specific antibody with a magnetic marker, a second sprayer that sprays a reagent containing a specific antibody with a fluorescent marker, and a reagent containing a specific antibody with a dye marker. and a third atomizer.
  • a plurality of sprayers spray the reagents onto the saliva adhered to the test rod 20, respectively.
  • Multiple nebulizers for example, nebulize reagents at the same time or at different times.
  • the inspection device 100 can switch between two types or three types of marker-attached specific antibodies 40 and mix them with saliva.
  • the inspection apparatus 100 performs the inspection using three types of specific antibodies 40 with markers when receiving a request to perform an inspection with higher accuracy from the operator.
  • the inspection apparatus 100 may perform the inspection using two types of marker-equipped specific antibodies 40 when receiving a request from the operator to perform the inspection at a higher speed.
  • the attachment section 120 may have a drip section that drips a reagent containing a specific antibody with a marker onto saliva adhered to the test rod 20 .
  • the attachment section 120 may have a plurality of dropping sections for dropping reagents corresponding to the types of specific antibodies with markers. A plurality of dropping units drop reagents at the same timing or different timings, for example.
  • the attaching section 120 can mix the saliva of the subject 10 and the reagent even by using such a dropping section.
  • the detection unit 160 detects the second light in S56.
  • the detection unit 160 moves the conjugates 50.
  • a second light having an intensity equal to or higher than the threshold may be detected even while the light is being turned on.
  • the control unit 180 applies a magnetic field to move the combined body 50 in S55, and moves in the predetermined region in S56.
  • the detection unit 160 may be caused to detect the intensity of the second light received from the combined body 50 that is present.
  • the control unit 180 causes the detection unit 160 to start the detection operation immediately after the magnetic field application unit 150 applies the magnetic field.
  • the determination unit 181 determines that the virus 30 is contained in the saliva of the subject 10 when the intensity of the second light detected by the detection unit 160 exceeds the threshold even when the conjugate 50 is moving. can be determined.
  • the determination unit 181 can also determine whether the virus 30 is contained in the saliva of the subject 10 based on the temporal change in the intensity of the second light detected by the detection unit 160 .
  • the determining unit 181 can determine that the virus 30 is contained in the saliva of the subject 10 according to the temporal change in the intensity of the second light.
  • the detection unit 160 may have an imaging unit such as a CCD and take an image of a predetermined area. Since the magnetic field applying unit 150 applies a magnetic field to move the combined body 50, the bright spot of the second light generated from the combined body 50 moves with time in a predetermined region. Therefore, the detection unit 160 captures a moving image of the movement of the plurality of bright spots of the second light.
  • the determination unit 181 can determine whether or not the plurality of combined bodies 50 are moving by image analysis of such moving image data. Therefore, the determination unit 181 can determine that the virus 30 is contained in the saliva of the subject 10 according to the temporal movement of the bright spot of the second light.
  • the magnetic field application unit 150 applies the magnetic field and the detection unit 160 starts the detection operation, so that the testing device 100 can more quickly test whether the subject 10 is infected with the virus 30 or not. can.
  • the inspection apparatus 100 irradiates the predetermined region with the first light in S54 and then applies the magnetic field to the liquid mixture in S55
  • the inspection apparatus 100 is not limited to this.
  • the inspection apparatus 100 may irradiate the predetermined region with the first light after applying the magnetic field to the liquid mixture to move the conjugate 50 in S55.
  • the operation in this case is an operation in which S54 and S55 in the operation flow in FIG. 5 are interchanged.
  • test rod 20 may be pre-attached with a reagent containing the specific antibody 40 with a marker.
  • the saliva and the reagent are mixed. Therefore, by using the test rod 20 to which the reagent containing the marker-attached specific antibody 40 is previously attached to the attachment surface 21, the subject's saliva and the reagent can be mixed. In this case, furthermore, the application section 120 operates to adhere the reagent, so that the subject's saliva and the reagent can be mixed more reliably.
  • the attachment section 120 does not need to operate.
  • the operations of S51 and S52 in FIG. 5 can be omitted, and the inspection time can be shortened.
  • the inspection apparatus 100 may not be provided with the adhesion section 120 . In this case, the manufacturing cost of the inspection device 100 can be reduced.
  • the inspection apparatus 100 In the inspection apparatus 100 according to the present embodiment described above, an example in which the test rod 20 is provided with the groove 23 and the observation area 24 has been described.
  • a plurality of grooves 23 and observation regions 24 may be provided on the test rod 20 . It is desirable that the subject 10 adheres saliva to a plurality of regions corresponding to the plurality of grooves 23 and the observation region 24 of the test rod 20 .
  • the attaching section 120 attaches a reagent to a plurality of areas to which saliva has adhered. Thereby, the inspection apparatus 100 can sequentially inspect a plurality of areas.
  • the attaching unit 120 attaches the same reagent to multiple regions. Thereby, the inspection apparatus 100 can efficiently perform the same inspection multiple times. Also, the attachment section 120 may attach a different reagent to each region. For example, if different reagents are used for each virus 30 to be detected, the testing device 100 can efficiently sequentially perform tests for each virus 30 .
  • the magnetic field applying unit 150 applies a magnetic field to collect the combined bodies 50 in a local area
  • the present invention is not limited to this.
  • a reagent containing the marker-attached specific antibody 40 may be attached in advance to the local observation area 24 of the test rod 20 .
  • Such a test rod 20 will be described below.
  • FIG. 6 shows a modification of the test rod 20 according to this embodiment.
  • the same reference numerals are given to the parts that are substantially the same as the operation of the test rod 20 according to the present embodiment shown in FIG. 4, and overlapping explanations are omitted.
  • a modified test rod 20 is provided with a groove 23 in an observation area 24 .
  • a water retention agent 25 is provided in the groove 23 .
  • a reagent containing a marker-attached specific antibody 40 is attached to the water retention agent 25 .
  • the subject 10 attaches the saliva of the subject 10 to the attachment surface 21 of the test rod 20, the saliva and the reagent are mixed in the observation area 24.
  • the bonding part 120 and the magnetic field application part 150 do not need to operate because the combined body 50 is generated in the observation area 24.
  • FIG. for example, the operations of S51, S52, and S55 in FIG. 5 can be omitted, and the inspection time can be shortened.
  • the inspection apparatus 100 may not be provided with the adhesion section 120 and the magnetic field application section 150 . In this case, the manufacturing cost of the inspection device 100 can be reduced.
  • the attachment portion 120 may be used to attach the marker-equipped specific antibody 40 to the observation area 24 .
  • the application section 120 may further include a sprayer for spraying a solution such as a buffer liquid or water onto the observation area 24 .
  • the buffer solution is, for example, PBS (phosphate buffered saline) or the like.
  • FIG. 7 shows a modification of the operation flow of the inspection apparatus 100 according to this embodiment.
  • the same reference numerals are given to the operations that are substantially the same as the operation flow of the inspection apparatus 100 according to the present embodiment shown in FIG. 5, and overlapping explanations are omitted.
  • the operation flow of FIG. 7 is the operation after the saliva of the subject 10 is attached to the test rod 20 shown in FIG.
  • control section 180 controls the moving section to move the attaching section 120 to a position corresponding to the insertion opening 110 (S51). Then, the control unit 180 controls the sprayer to spray a solution such as a buffer solution or water onto the observation area 24 (S71). As a result, the marker-attached specific antibody 40 other than the conjugate 50 can be washed out of the observation region 24 . Next, the control unit 180 controls the attachment unit 120 to spray the reagent containing the specific antibody 40 with the marker onto the observation area 24 of the test rod 20 (S72). This makes the production of the conjugate 50 more reliable.
  • control section 180 controls the vibration section 130 to vibrate the test rod 20 (S53). Note that the control section 180 may vibrate the test rod 20 also before and after the operation of S71. Then, the control unit 180 controls the sprayer to spray a solution such as a buffer solution or water onto the observation area 24 (S73). As a result, the marker-attached specific antibody 40 other than the conjugate 50 is washed out of the observation region 24 . As described above, when virus 30 is contained in saliva, multiple conjugates 50 can be generated in observation area 24 .
  • control unit 180 controls the light source unit 140 to irradiate the mixture of saliva and reagent with the first light (S54).
  • the light source unit 140 irradiates the observation area 24 .
  • control unit 180 controls the detection unit 160 to detect the intensity of the second light received from the observation area 24 due to the irradiation of the first light (S56).
  • the determination unit 181 determines whether the virus 30 is contained in the saliva of the subject 10 based on the detected light intensity (S57).
  • the inspection device 100 can generate the conjugate 50 in the local observation area 24 of the test rod 20, so that the operation of collecting the conjugate 50 is omitted for inspection. can do.
  • the application unit 120 sprays the reagent locally on the observation region 24, the amount of reagent used can be reduced.
  • the inspection device 100 does not need to use a specific antibody with a magnetic marker to collect the conjugate 50.
  • the reagent includes a fluorescent marker-attached specific antibody as the marker-attached specific antibody 40, and the detection unit 160 detects fluorescence emitted from the fluorescent substance as the second light.
  • the reagent may include a dye marker-attached specific antibody as the marker-attached specific antibody 40, and the detection unit 160 may detect reflected light and/or transmitted light of the first light as the second light.
  • the reagent may include a fluorescent marker-attached specific antibody and a dye marker-attached specific antibody as the marker-attached specific antibody 40 .
  • the reagent may contain a specific antibody with a magnetic marker to detect the virus 30.
  • a magnetic material is magnetized by a magnetic field applied from the outside. The more magnets are gathered, the more magnetism is generated. Therefore, the inspection apparatus 100 may apply the magnetic field by the magnetic field applying section 150 to the observation area 24 instead of the irradiation of the first light in S54.
  • the detection unit 160 detects the generated magnetism.
  • the detector 160 has a magnetic sensor. Thereby, the inspection device 100 can magnetically detect the combined body 50 .
  • the magnetic field applying section 150 may apply an alternating magnetic field to the observation region 24 . Then, the magnetic field applying section 150 may sweep the frequency of the alternating magnetic field.
  • a magnetic body has a magnetic resonance frequency peculiar to the magnetic body. Therefore, when the magnetic field applying unit 150 sweeps the frequency of the alternating magnetic field, the magnetism generated by the magnetic material fluctuates greatly in the vicinity of the magnetic resonance frequency.
  • the determination unit 181 can determine that the saliva of the subject 10 contains the virus 30 in response to the detection of such a change by the detection unit 160 .
  • the inspection apparatus 100 performs magnetic detection inspection of the conjugate 50 using a specific antibody with a magnetic marker, and optical detection of the conjugate 50 using a specific antibody with a fluorescent marker and/or a specific antibody with a dye marker. and may be used together.
  • a reagent containing the specific antibody 40 with a marker adhered to the local observation area 24 of the test rod 20 but the present invention is not limited to this.
  • the localized observation area 24 of the test rod 20 may be coated with reagents containing specific antibodies without markers 42 .
  • a specific antibody without a marker has multiple antibodies 41 arranged around it and does not have a marker 42 inside.
  • the markerless specific antibody binds to the spike protein-attached antigen 31 of the virus 30 to form a conjugate 50 in the same way as the marker-attached specific antibody 40 . Even if such a specific antibody is used, the inspection apparatus 100 attaches a reagent containing the specific antibody 40 with the marker to the observation area 24 in S72, so that the conjugate 50 without the marker 42 is specific with the marker.
  • Antibodies 40 can be added.
  • the plurality of inspection devices 100 can detect viruses 30 by using a common test rod 20 having a specific antibody without markers. is contained in the saliva of the subject 10 or not.
  • the reagent contains a specific antibody with a dye marker as the specific antibody with a marker 40
  • the reagents may contain different types of specific antibodies with dye markers.
  • the reagent is, for example, a specific antibody with a first dye marker to which an antibody and a first dye of a first color are bound, and a second dye of a second color different from the antibody and the first color are bound. and a second dye-marker-attached specific antibody.
  • the first light is preferably light containing the first color light and the second color light.
  • the first light is white light.
  • the reflected light of the first color light and the second color light or the first The transmitted light in which the intensity of the light of the color and the light of the second color is reduced becomes the second light.
  • the first color is red
  • the second color is blue
  • the first light is white light or light having a light intensity distribution similar to white light.
  • the first light is, for example, sunlight.
  • the first light may be light emitted from the light source unit 140 such as an LED, a fluorescent lamp, a lamp, or the like.
  • the light source unit 140 may be omitted.
  • the light source unit 140 is an LED, a fluorescent lamp, a lamp, or the like, the first light may be continuously turned on.
  • the light source unit 140 may be a light source that functions as a room light that illuminates the room.
  • the light source unit 140 may be a strobe included in the imaging unit.
  • the detection unit 160 detects red light and blue light. Reflected light containing the light or transmitted light in which the intensity of the irradiated red light and blue light is reduced is detected as the second light.
  • the determination unit 181 determines whether the virus 30 is a subject or not according to the fact that the second light generated from one conjugate 50 includes the light of the first color and the light of the second color. may be determined to be contained in the saliva of For example, the determination unit 181 determines whether or not the optical microscope image of one combined body 50 includes pixels of the first color and pixels of light of the second color.
  • the determination unit 181 numerically processes the captured images of the plurality of conjugates 50 to determine whether one conjugate 50 contains the first dye marker-attached specific antibody and the second dye marker-attached specific antibody. It may be determined statistically whether or not there is.
  • the detection unit 160 has an imaging unit such as a camera and a CCD, and images the second light generated from a predetermined area. Then, the determination unit 181 determines that pixels of the first color and pixels of the second color are included among the pixels of the imaging region in which the combined body 50 is captured from the captured image captured by the imaging unit. Accordingly, it is determined that the virus 30 is contained in the subject's saliva.
  • the determination unit 181 extracts the imaging region in which the plurality of combined bodies 50 are captured by extracting boundaries from the captured image.
  • particles to which several specific antibodies with dye markers of the same color are bound may be mixed in the reagent, and such particles may be extracted as noise by boundary extraction. .
  • the determination unit 181 calculates the standard deviation of the color gamut of pixels for each extracted region. For example, a region of a particle to which several red dye-marked specific antibodies are bound results in mostly red pixels, so the standard deviation of the red color gamut tends to be smaller. Similarly, the area of the particle to which several blue dye-marked specific antibodies are bound becomes almost blue pixels, so the standard deviation of the blue color gamut is smaller.
  • the conjugate 50 contains the first dye-marker-attached specific antibody and the second dye-marker-attached specific antibody, it includes red pixels and blue pixels, resulting in variations in color dispersion.
  • the standard deviation of the red color gamut is larger than the standard deviation of the particles to which the specific antibody with the red dye marker is bound, and the standard deviation of the blue color gamut is the specific antibody with the blue dye marker. is greater than the standard deviation of the bound particles. Therefore, in the red color gamut and/or the blue color gamut, the determining unit 181 determines whether the virus 30 may be determined to be contained in the subject's saliva.
  • the determination unit 181 may further calculate the probability density distribution of the standard deviation for each color gamut. By using the probability density distribution, the determination unit 181 can more accurately determine whether or not the conjugate 50 having a plurality of dyes is present without depending on the number of particles.
  • the determination unit 181 determines that the virus 30 is contained in the subject's saliva, for example, when the half width of the probability density distribution is equal to or greater than the threshold.
  • the determining unit 181 determines that the virus 30 is contained in the subject's saliva when the integrated value of the probability density distribution (having a predetermined standard deviation or more) within a predetermined range is equal to or greater than the threshold.
  • the determination unit 181 may use Euclidean distance, Mahalanobis sail distance, or the like.
  • Euclidean distance when a population is uniformly distributed in a circle on two-dimensional coordinates, it is possible to determine whether or not a sample belongs to the population by calculating the Euclidean distance to an unknown sample and performing threshold discrimination.
  • the population has, for example, an elliptical distribution, so by correcting these and assuming it to be a circular population (coordinate transformation), obtaining the Mahalanobis sail distance and comparing it with a threshold, You can determine whether you belong to the population or not.
  • the testing device 100 can test whether or not the virus 30 is contained in the saliva of the subject 10 even when using different types of reagents containing specific antibodies with dye markers.
  • the inspection device 100 may use reagents containing different types of dye-marker-attached specific antibodies and magnetic-marker-attached specific antibodies to move the conjugate 50 to a predetermined region.
  • the reagent may contain only one type of specific antibody with a dye marker, in which the antibody is bound to a monochromatic dye.
  • the determining unit 181 calculates the size of the extracted region, and acquires the distribution of the numbers with respect to the size of the region (particle size).
  • FIG. 8 shows an example of the distribution of the number of particles with respect to the particle size of particles detected by the inspection device 100 according to this embodiment.
  • FIG. 8 shows an example of the distribution when the conjugate 50 indicated as Spike is formed and the distribution when the conjugate 50 indicated as Control is not formed.
  • the determination unit 181 determines whether the virus 30 is contained in the subject's saliva in response to the integration value of the predetermined range (the range in which the particle size is larger than the predetermined value) in the acquired distribution. It can be determined that there are
  • the determination unit 181 may normalize the distribution of the number of particles with respect to the particle size.
  • FIG. 9 shows an example of normalizing the distribution of the number of particles with respect to the particle size of particles detected by the inspection apparatus 100 according to this embodiment.
  • normalization is normalization by the number of particles of a certain specific particle size (a ⁇ X ⁇ b).
  • FIG. 9 shows an example of the distribution of FIG. 8 normalized by the number of particles where 0 ⁇ X ⁇ 100.
  • Example of Forming Combined Body 50 In the present embodiment described above, it has been described that the virus 30 and the marker-attached specific antibody 40 bind to form the conjugate 50 .
  • a simulated coronavirus sample A
  • the conjugate 50 is actually formed.
  • Sample A is a sample with a particle diameter of 10 nm, in which an antigen (delta-type novel coronavirus spike protein, virus receptor binding region) is bound to gold beads with a diameter of 10 nm to make a pseudo-coronavirus.
  • Sample B is a sample in which a monoclonal antibody is immobilized on a marker with a diameter of 335 nm to obtain a marker-attached specific antibody 40 .
  • FIG. 10 shows an electron microscope image after mixing such sample A and sample B in a micro test tube (1.5 mL) and stirring for several seconds.
  • FIG. 10 shows an example in which the spike protein-attached antigen 31 of the virus 30 is bound to the antibody 41 of the marker-attached specific antibody 40 according to this embodiment.
  • FIG. 10 shows that a plurality of samples A are bound to one sample B.
  • FIG. 11 shows a first example of a comparison target.
  • FIG. 11 shows an electron microscope image after mixing sample A' and sample B in a micro test tube (1.5 mL) and stirring for several seconds.
  • sample A' is a sample in which a control protein (antigen) unrelated to the spike protein is bound to gold beads. It can be seen in FIG. 11 that very few samples A' are bound to one sample B.
  • FIG. 11 shows a control protein (antigen) unrelated to the spike protein is bound to gold beads.
  • FIG. 12 shows a second example for comparison.
  • FIG. 12 shows an electron microscope image after mixing sample A and sample B' in a micro test tube (1.5 mL) and stirring for several seconds.
  • sample B' is a sample conjugated with a specific monoclonal antibody against a protein unrelated to the spike protein.
  • FIG. 12 it can be seen that there is no sample A bound to one sample B'.
  • FIG. 13 shows a third example for comparison.
  • FIG. 13 shows an electron microscope image after mixing sample A' and sample B' in a micro test tube (1.5 mL) and stirring for several seconds.
  • FIGS. 10 to 13 it can be seen that antigen 31 with spike protein of virus 30 binds to antibody 41 of specific antibody 40 with marker.
  • FIG. 14 shows an example in which the combined body 50 according to the present embodiment is formed in an optical microscope image of approximately 4000 times.
  • FIG. 14(A) shows an optical microscope image after mixing sample A' and sample B described in FIG.
  • FIG. 14B shows an optical microscope image after mixing the sample A and the sample B described in FIG.
  • sample B marker-attached specific antibody 40
  • a mixture of marker-attached specific antibody 40 containing red dye beads and marker-attached specific antibody 40 containing blue dye beads was used. Since the reagent is a liquid, it is thick and out of focus, and there are portions where the particle diameter of the sample B appears to be large due to aberration.
  • Fig. 14(A) shows that small-diameter particles are scattered and the conjugate 50 is not formed.
  • FIG. 14(B) particles with a diameter exceeding 5 ⁇ m are observed, indicating that the conjugate 50 is formed.
  • FIG. 14(A) individual red and blue dye beads are scattered. I was able to confirm that there was. As described above, it was found that the conjugate 50 can be formed by mixing the pseudo virus 30 and the marker-attached specific antibody 40 .

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Abstract

Un procédé d'inspection comprend : une étape consistant à mélanger la salive d'un sujet et un réactif comprenant un anticorps spécifique fixé à un marqueur ; une étape consistant à exposer un mélange liquide de la salive et du réactif à une première lumière d'une longueur d'onde prédéterminée ; une étape consistant à détecter l'intensité d'une seconde lumière générée par l'exposition à la première lumière ; et une étape consistant à déterminer, en fonction de l'intensité lumineuse détectée, si des virus sont contenus dans la salive du sujet, le réactif comprenant au moins un anticorps spécifique parmi un anticorps spécifique fixé à un marqueur fluorescent ou un anticorps spécifique fixé à un marqueur de coloration.
PCT/JP2023/005542 2022-02-22 2023-02-16 Procédé d'inspection et dispositif d'inspection WO2023162859A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004267202A (ja) * 2003-02-21 2004-09-30 Toyama Univ アミラーゼ活性測定キット
JP2011528786A (ja) * 2008-07-18 2011-11-24 アルトシュル、ランディス−リサ 唾液の無侵襲解析方法及び装置
JP2017536084A (ja) * 2014-09-30 2017-12-07 ジーイー・ヘルスケア・ユーケイ・リミテッド 口腔癌バイオマーカーの検出に関連する方法及びデバイス
WO2022036330A1 (fr) * 2020-08-14 2022-02-17 Verevas, Inc. Dosage d'agglutination de lieu d'intervention de coronavirus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004267202A (ja) * 2003-02-21 2004-09-30 Toyama Univ アミラーゼ活性測定キット
JP2011528786A (ja) * 2008-07-18 2011-11-24 アルトシュル、ランディス−リサ 唾液の無侵襲解析方法及び装置
JP2017536084A (ja) * 2014-09-30 2017-12-07 ジーイー・ヘルスケア・ユーケイ・リミテッド 口腔癌バイオマーカーの検出に関連する方法及びデバイス
WO2022036330A1 (fr) * 2020-08-14 2022-02-17 Verevas, Inc. Dosage d'agglutination de lieu d'intervention de coronavirus

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