WO2014058064A1

WO2014058064A1 - Inspection system and inspection method

Info

Publication number: WO2014058064A1
Application number: PCT/JP2013/077811
Authority: WO
Inventors: みつ子関; 友野　潤; 重彦宮本
Original assignee: 学校法人日本大学; 株式会社カネカ
Priority date: 2012-10-12
Filing date: 2013-10-11
Publication date: 2014-04-17
Also published as: JPWO2014058064A1

Abstract

An inspection system and an inspection method for analyzing and investigating the presence or absence of a plurality of types of detection objects in an object to be inspected, the system comprising: an inspection device that has a plurality of recessed portions to which the object to be inspected is applied, and previously holds, within the respective recessed portions, reagents corresponding to the respective detection objects and each including a coloring agent that takes on coloration or changes color due to the presence of the detection object; an image capturing means for capturing images of the plurality of recessed portions of the inspection device; and a computer provided with a storage means for storing the arrangement of the plurality of recessed portions of the inspection device and the detection objects in the respective recessed portions, and a determination means for comparing the coloration or color change pattern of all of the plurality of recessed portions the images of which are captured by the image capturing means and the arrangement of the recessed portions in the storage means, and determining the presence or absence of each of the detection objects.

Description

Inspection system and inspection method

The present invention relates to an inspection system and an inspection method for analyzing and investigating the presence or absence of a plurality of types of detection targets for an inspection object.

Conventionally, in various fields such as medical care, life science, food analysis, and environmental measurement, various substances (for example, nucleic acids) in an object to be inspected are analyzed and investigated. And based on the results obtained there, it has been used for subsequent treatment and treatment policy decisions.

However, depending on the detection target, since only a trace amount is contained in the inspected object, detection may be performed after the detection target is amplified or propagated. For example, in the medical and life science fields, a method of performing nucleic acid amplification by a PCR (Polymerase Chain Reaction) method has been widely used in the past for analyzing and investigating nucleic acids in a test object.

Then, after performing nucleic acid amplification by the PCR method, as a method of confirming whether or not the nucleic acid to be detected exists in the test object, a probe is attached to some solid support, and the corresponding nucleic acid is A method of detecting is proposed (see Patent Document 1).

However, the PCR method requires a complicated temperature control program for heating and cooling to an optimum temperature, and a large dedicated device is required to perform nucleic acid amplification by the method. Therefore, a LAMP (Loop-Mediated Isolation Amplification) method that can amplify a nucleic acid only by keeping it at a constant temperature around 65 ° C. has been proposed (see Patent Document 2). If the LAMP method is used, it can be carried out as long as there is an instrument that can keep a mixture of an object to be inspected and a dedicated reagent, such as a pot, at around 65 ° C. This is a useful method from the viewpoint of simplifying the process.

However, for example, even if the presence or absence of nucleic acids to be detected can be easily determined in the field, the substance information obtained there is analyzed, and certain results useful for subsequent treatment and treatment policy determination are accurately grasped. Requires a high level of expertise and requires time and effort. Those who do not have specialized knowledge need to send the inspection object to a specialized facility and obtain the analysis results at a later date. For example, in the medical field, it is necessary to obtain information on the patient's pathology as soon as possible and use it accurately in the formulation of the next treatment policy. However, there are detection technologies for determining the presence of various target substances. However, there was no way to easily analyze much of the material information obtained there without specialized knowledge on the spot.

JP 2012-105663 A Japanese Patent No. 3974441

In view of the circumstances as described above, the object of the present invention is an inspection that can analyze the inspection object on-site and can easily and quickly grasp the analysis result easily without specialized knowledge. To provide a system and an inspection method.

That is, the gist of the present invention is as follows.
[1] An inspection system for analyzing and investigating the presence / absence of a plurality of types of detection targets for an inspection object, having a plurality of recesses to which the inspection object is applied, An inspection apparatus that holds in advance a reagent that includes a coloring agent that changes color due to the presence of the detection target, and an imaging unit that images the plurality of recesses of the inspection apparatus; Storage means for storing the array of the plurality of recesses and the detection target of each recess of the inspection apparatus, and a coloration or discoloration pattern of the whole of the plurality of recesses imaged by the imaging means, and an array of the recesses of the storage means An inspection system comprising: a computer including a determination unit that determines whether or not each detection target is present by comparing
[2] The inspection system according to [1], wherein the reagent contains a reaction promoting substance that amplifies or proliferates the detection target.
[3] The inspection system according to [1] or [2], wherein the detection target is a nucleic acid,
[4] The inspection system according to any one of [1] to [3], wherein the coloring agent is a leuco dye.
[5] The inspection system according to any one of [1] to [4], further comprising temperature adjusting means for promoting a reaction between the test object applied to the recess and the reagent.
[6] The imaging unit includes a camera that images the plurality of recesses, a communication control unit that transmits and receives information to and from the computer, and a client terminal that includes an information display unit. The imaging data comprising a color or a discoloration pattern is transmitted from the client terminal to the computer, and the determination result received from the computer is displayed on the information display means of the client terminal according to any one of [1] to [5] Inspection system,
[7] The inspection system according to [6], wherein the client terminal is a portable terminal having a wireless communication control unit as the communication control unit, and is connected to the computer via a wireless communication network.
[8] An inspection method for analyzing and investigating the presence or absence of a plurality of types of detection targets for an inspection object, having a plurality of recesses to which the inspection object is applied, and corresponding to each detection object inside each recess An inspection apparatus that holds in advance a reagent that is a reagent and contains a coloring agent that changes color or changes color due to the presence of the detection target, an imaging unit that images the plurality of recesses of the inspection apparatus, and the inspection Comparing the arrangement of the plurality of recesses of the apparatus and the storage means for storing the detection target of each recess, and the coloration or discoloration pattern of the whole of the plurality of recesses imaged by the imaging means and the arrangement of the recesses of the storage means And a computer having a determination means for determining the presence or absence of a detection target, a procedure for applying an object to be inspected to the plurality of recesses of the inspection apparatus, and a coloration or coloration of the plurality of recesses by the imaging means. A procedure for imaging a discoloration pattern, a procedure for the computer to determine the presence or absence of each detection target by comparing the coloration or discoloration pattern imaged by the imaging means and the arrangement of the concave portions of the storage means, and Inspection method,
[9] The imaging unit includes a camera that images the plurality of recesses, a communication control unit that transmits and receives information to and from the computer, and a client terminal that includes an information display unit. , A procedure for applying an object to be inspected, a procedure for the client terminal to image a coloration or discoloration pattern of the whole of the plurality of recesses by the camera, and the color or color of the client terminal captured by the camera A procedure for transmitting imaging data comprising a discoloration pattern to the computer, and the computer compares the coloration or discoloration pattern of the imaging data received from the client terminal with the array of recesses in the storage means, A procedure for determining the presence or absence of the computer, a procedure for the computer to transmit a determination result to the client terminal, Serial client terminal, inspection method of the additional level [8], wherein the procedure for displaying the determination result received from the computer to the information display means,
It is.

According to the present invention as described above, an object to be inspected is applied to a plurality of concave portions of the inspection apparatus, and is colored or discolored depending on the presence or absence of a detection target, or from all the concave portions that are not colored or discolored. The coloration or discoloration pattern to be detected is imaged by the imaging means, and the presence or absence of each detection target can be analyzed by comparing the captured coloration or coloration pattern with the array of the concave portions of the storage means. If there is a portable computer and camera, the above-mentioned coloration or discoloration pattern is imaged by an imaging means with a simple operation as if reading a bar code or QR code (registered trademark) in the field, and the captured pattern data is obtained. By making a computer judgment, accurate analysis and survey results can be obtained quickly and easily without specialized knowledge. Selection of dosing drugs in, it is possible to help immediately policy decisions. In addition, since it is possible to realize the determination by recognizing the coloration or discoloration pattern with a relatively small amount of calculation, it can be realized without using a special computer, and the determination is made by the computer of the user who installed the software. Therefore, if the computer is brought to the site, the result can be obtained immediately.

Also, if the reagent held in the recess contains a reaction promoting substance that amplifies or proliferates the detection target, for example, the detection target such as nucleic acid can be analyzed quickly.

In addition, if the coloring agent is a leuco dye, it produces a clear coloration or discoloration under visible light that can be recognized by a normal camera, so that clear pattern data can be obtained and reliable analysis can be performed by a computer based on this. Can be made.

Also, if a temperature control means for promoting the reaction between the sample and the reagent placed in the recess is provided, the reaction can be accelerated and work can be performed efficiently, and the system is suitable for on-site use.

Further, the imaging device is a client terminal including a camera that images a plurality of recesses, a communication control unit that transmits and receives information to and from the computer, and an information display unit, and a coloration or discoloration pattern of the entire recess captured by the camera If the system is configured to transmit imaging data comprising a client terminal to a computer and display the determination result received from the computer on the information display means of the client terminal, the computer including the determination program is not carried on site. If there is a general-purpose client terminal at or near the site, imaging data can be transmitted from the client terminal to the remote computer, and the determination result can be obtained immediately on the spot.

In addition, if the client terminal is a portable terminal having a wireless communication control unit as a communication control unit and is connected to the computer via a wireless communication network, for example, a camera, a communication control unit, and an information display unit are provided. A portable terminal such as a mobile phone, a smart phone, a tablet computer, a PDA, or a notebook personal computer is carried along with the inspection device to the site, and imaging data is transmitted to the remote computer, and the determination result can be obtained immediately at the site. it can.

Schematic which shows the inspection apparatus used for the inspection system which concerns on typical embodiment of this invention. Explanatory drawing which similarly shows the procedure until it acquires the imaging data of the inspection method which concerns on typical embodiment. Explanatory drawing which similarly shows the procedure after the imaging data acquisition of the inspection method which concerns on typical embodiment. The block diagram which shows the structure of the computer similarly used for a test | inspection system. Explanatory drawing which shows the modification of embodiment of this invention. The flowchart which shows the test | inspection procedure by a test | inspection system. The block diagram which shows the structure of the computer used for the test | inspection system of a modification. The flowchart which shows the test | inspection procedure by the test | inspection system of a modification similarly.

The inspection system according to the present invention analyzes and investigates the presence or absence of a plurality of types of detection targets for an inspection object. As shown in FIG. 2, a plurality of concave portions 10 for applying the inspection object A by a predetermined amount are provided. And at least a computer 3 including an imaging unit 2, a storage unit, and a determination unit. The purpose of the test to which the present invention can be applied includes not only genetic testing for nucleic acids and immunological testing for proteins, but also environmental analysis such as soil and air, water quality testing, food analysis, etc. It is not limited to these. Examples of genetic tests include single nucleotide polymorphisms and infectious disease diagnosis.

As the inspection object, various inspection objects corresponding to these purposes can be used, regardless of whether they are solid or liquid. Examples include excrement collected from humans and livestock, sputum, throat swab, blood, plasma, serum, urine, tissues, cells, clinical, non-clinical samples, water samples for water quality tests, food, soil, etc. However, it is not limited to these. When the detection target contained in these inspected objects is extremely small, for example, if the detection target is a microorganism such as a bacterium or virus, an inspection device is placed in a thermostatic chamber or a CO ₂ incubator to promote growth, If present, nucleic acid amplification may be promoted by various nucleic acid amplification means.

As the nucleic acid amplification means when the test object is a nucleic acid, known nucleic acid amplification means such as a PCR (Polymerase Chain Reaction) method can be widely used. However, it is preferable to use isothermal nucleic acid amplification means capable of performing nucleic acid amplification under isothermal conditions, for example, LAMP method, ICAN (Isothermal and Chimeric primary-Amplified of Nucleic Acids) method, PALSAR, etc. (Probe Alternation Link Self-Assembly Reaction) method and SmartAmp (Smart Amplification Process) method, but not limited thereto.

Examples of detection targets include nucleic acids, proteins, microorganisms (bacteria, viruses, etc.), parasites, physiologically active substances, lipids, pharmaceuticals, blood cells, genes, various substances in soil, water and air (residual chlorine, nitrite ions) Nitrite nitrogen, COD, ammonium, phosphoric acid, potassium, etc.), heavy metals, food additives, residual agricultural chemicals, and the like, but are not limited thereto.

In the following embodiments, a system for performing remote communication between a computer 3 and a portable computer terminal (mobile terminal 5), which is a client terminal 4, will be described. However, the client terminal 4 is omitted, and imaging means, storage Needless to say, a computer having a means, a determination means, a display means, etc. may perform from imaging to display of the determination result (modified example described later). In this case, it is possible to bring the computer into the field as a portable type. Of course, the client terminal 4 may be a stationary computer terminal instead of a portable computer (mobile terminal 5). In addition, the computer 3 can be a portable computer similar to the client terminal 4. Of course, a separate camera device that can be connected to the client terminal 4 or the computer 3 may be used as the imaging means.

As shown in FIG. 1A, the representative embodiment of the inspection apparatus 1 is provided with a plurality of bottomed recesses 10 that are open on the upper surface 12a of the plate-like main body 12 in the vertical and horizontal directions. The color of the plate-like main body 12 is set according to the type of the object to be inspected, the reagent, and the coloring agent so that the coloration or discoloration pattern by the coloring agent appears clearly, and can be any of transparent to translucent color, white or black A single color or the like is preferable, but is not particularly limited. The thickness from the upper surface 12a to the lower surface of the plate-like main body 12 is not particularly limited. The material of the inspection apparatus 1 is not particularly limited, and may be selected according to an object to be inspected and a reagent such as various synthetic resins, ceramics, and glass. However, when it is assumed that the inspection apparatus 1 is heated or cooled, a material that can adapt to these temperature changes is selected.

The number and arrangement method of the recesses 10 are not particularly limited, and may be a single row as long as it is two or more. In addition, a so-called 96-hole, 384-hole plate, and multiple tube, which are widely used in the life science research area, may be used as the inspection apparatus. In this example, the shape of each concave portion 10 is an example in which a cylindrical storage space having a curved bottom portion is formed. However, the shape of each concave portion 10 is not limited to such a shape. Various shapes such as a conical space may be used. In addition, it is good also as a shape which provided the cell or area by the wall or the partition. In addition, a section for extracting and purifying nucleic acids and proteins, a section for performing nucleic acid amplification, and a flow path for delivering an object to be inspected to the recess 10 may be provided. In order to discriminate the coloration or discoloration pattern, it is preferable that the shape of each recess 10 is the same shape and size, but in order to recognize the reference point at the time of determination, the shape of one or more recesses In particular, it is also preferable to make the opening shape and dimensions different from others. In addition, in order to prevent evaporation of the inspection object and reagent and mixing from other recesses, a structure may be formed in which a lid can be formed before and after applying the inspection object. As such a structure, for example, a mode in which the lid is covered with a sticker having adhesive force can be given. As another embodiment, for example, when a larger volume of sample is required for a test such as a culture solution or environmental analysis, a plurality of samples can be individually held as shown in FIG. An embodiment in which the tube 13 is used and the tube 13 is set in the tube rack 14 may be employed. Other than that, for example, as shown in FIG. 1C, a dedicated inspection chip 15 may be prepared as the inspection apparatus 1. The size of the inspection chip 15 is not particularly limited as long as it is a size that can be imaged by an imaging unit described later. The depth of the recess 10 at this time is not particularly limited as long as imaging is possible. However, if the depth is too deep, the analysis accuracy is adversely affected by the influence of shadows, and is preferably 10 cm or less. In any of the embodiments, the reference mark 16 on the upper surface of the inspection apparatus 1 and the concave portion (for example, positive control) serving as a reference are used to arrange the specimen and the rotation angle, inclination, and distance of imaging data to be described later. One or two or more may be set in order to correct (size) by a known means. For the purpose of easily obtaining information on the object to be inspected and the type of analysis / investigation, a barcode 17 or a QR code (registered trademark) may be printed or pasted on the inspection apparatus 1. .

When applying the inspection object to each recess 10, a suitable applying device 60 may be used according to the amount of the inspection object applied to each recess 10 and the size of the recess 10. Specific examples include a micropipette, a measuring pipette, a syringe, a dropper, and the like. Basically, any instrument that can accurately apply an object to be inspected may be used, and the present invention is not limited thereto. However, for example, when there is only one type of object A to be inspected and there is almost no possibility that the reagents held in the respective recesses are mixed, or even if they are mixed somewhat, the inspection accuracy is not greatly affected. When a high degree of accuracy is not required for the amount of the inspection object A to be applied, for example, an extremely small inspection apparatus 1 is prepared, and the inspection object A is applied to all the concave portions at once by the applying tool 60. Such a mode may be sufficient. In addition, when high accuracy is not required for the amount of the inspection object to be applied, the inspection object can be simply applied by decantation or the like without using the applying device 60. Even if a high degree of accuracy is required for the amount of the inspection object A to be applied, the work efficiency may be improved by using a multi-channel micropipette. Various application methods are possible depending on the nature of the reaction and the form of the inspection apparatus 1, but the present invention is not limited thereto, and various methods may be selected according to the form of each inspection object and inspection apparatus.

The size of the recess 10 may be appropriately set according to the size of the inspection apparatus 1 itself and the amount of the inspection object to be applied. Basically, it is preferable that there is a separation distance between the adjacent recesses 10 so that the reagent and the test object are hardly mixed even if the test object overflows from the recesses 10. In this respect, a protrusion or an absorption pad that protrudes upward is provided at the edge of each recess, or a groove is provided between the recesses so that the inspection object overflows from the recess and does not easily enter the adjacent recess. It is also preferable. However, as described above, the inspection accuracy is not greatly affected even if the inspected objects A to be applied are mixed to some extent. For example, as shown in FIG. When the inspection object A is applied to each of the recesses, it is preferable that the distance between the recesses is short, and it is preferable that there are no protrusions, absorption pads, or grooves protruding upward at the edge of each recess.

In each recess 10, a reagent 11 that is a reagent corresponding to the detection target and that contains a coloring agent that changes color or changes color due to the presence of the detection target is held in advance. The reagent 11 preferably contains a reaction promoting substance in addition to the coloring agent.

The coloring agent is not particularly limited as long as it is colored or discolored due to the presence of the detection target, and may be colored or discolored by direct reaction with the detection target itself. It may be good, or it may be colored or discolored via a reaction accelerator described later. Examples of the coloring agent include an acid-base indicator that changes color or changes color depending on the acid dissociation constant of the object to be inspected, a leuco type dye that changes color or changes color when it reacts with the detection target, and other detection agents. These reagents are mentioned.

Basically, it is preferable to select a coloring agent that can detect such coloration or discoloration under visible light because it is simple, but it is necessary to increase the detection sensitivity for a small amount of detection target. Depending on various circumstances, for example, a fluorescent labeling reagent that can detect the coloration or discoloration by fluorescence emitted under excitation light may be used as a coloring agent.

Examples of acid-base indicators include picric acid, methyl violet, o-cresol red, thymol blue, 2,4-dinitrophenol, congo red, methyl orange, methyl yellow, bromophenol blue, bromocresol green, methyl red, litmus , Methyl purple, bromocresol purple, chlorophenol red, bromotinol blue, p-nitrophenol, neutral red, phenol red, p-naphtholphthalein, cresol red, thymol blue, phenolphthalein, thymolphthalein, alizarin yellow R, 1,3,5-trinitrobenzene and the like.

In the present invention, the leuco dye is a colorless or light-colored dye, and means a dye that changes its structure by a reaction with a developer or a physical stimulus such as light and exhibits coloration. For example, a leuco dye used for detecting a multi-stranded nucleic acid is based on the detection principle that a chromogenic leuco dye is added to the multi-stranded nucleic acid and colored by the interaction. Such a leuco dye is not particularly limited as long as it is substantially colored by the interaction with a nucleic acid. However, a triarylmethane dye, xanthene dye, quinoline dye, phenothiazine dye, phenoxy Examples include sazine dyes and mixtures thereof. Specific examples thereof include triarylmethane dyes such as methyl green, malachite green, crystal violet, pararosaniline, gentian violet B, gentian violet R, night blue, Victoria blue B, Victoria blue R, quinoline 4- ( Examples include leuco derivatives such as (p-dimethylaminostyryl) quinoline, phenothiazine phenothiazine, benzoyl leucomethylene blue, and phenoxazine phenoxazine, but are not limited thereto, as long as the leuco derivative is brought into contact with a nucleic acid. In addition, these leuco dyes can be used alone or in combination.
Among these, triarylmethane dyes are preferable, and derivatives of leuco dyes of crystal violet and gentian violet B are particularly preferable. Furthermore, in the present invention, the leuco dye can be converted from the coloring dye. For example, it can be converted into a leuco dye by reacting a nucleophilic agent with a triarylmethane-based coloring dye. In this case, even if a single triarylmethane dye is used, a leuco dye having a different structure can be obtained if the nucleophile used is changed. Examples thereof include one or more triarylmethane dyes selected from the group consisting of gentian violet, crystal violet, methyl green, malachite green, Victoria blue, pararosaniline and derivatives thereof, sulfite ion, hydrogen sulfite ion, nitric acid. One or more nucleophiles selected from the group consisting of ions, nitrite ions, cyanide ions, halide ions, nitrogen nucleophiles, sulfur nucleophiles, alkali metal alkoxides, alkali metal hydroxides and hydride nucleophiles. A reactant.

As the fluorescent labeling reagent, a fluorescein derivative, a rhodamine derivative, a Cy dye or other known ones may be used, and is not particularly limited. The method for binding these fluorescent labeling reagents to the detection target is not particularly limited, and for example, a method known in the technical field of each detection target may be used. Specifically, when a nucleic acid is fluorescently labeled, an intercalator method or a fluorescently labeled probe may be used, but is not limited thereto.

In addition, various detection reagents can be used depending on the detection target, for example, iodine solution for starch, malting or fructose, ferring reagent or benecto for glucose, silver nitrate aqueous solution for ammonia, and ammonia for chloride ion. In contrast, Nessler's reagent, indigo carmine solution for oxygen, and Coomassie blue reagent or bromophenol blue reagent for protein may be used.

When it is necessary to react with a coloring agent in the reaction process such as some chemical reaction, biological reaction, biochemical reaction, etc. or after the reaction at the time of detection, the chemical reaction, biological reaction, biochemical reaction Also included in the reagent 11 are reaction-promoting substances necessary for the above. For example, when the detection target is a nucleic acid, the reaction promoting substance includes reaction promoting substances such as various primers, DNA polymerase, substrate, and buffer.

The reagent 11 is held inside the recess 10 in an optimum state according to each detection reaction, for example, a solid, semi-solid (gel form, etc.), liquid, or the like. For example, a mode in which a reagent held in a powder or dry state is used after being diluted with a solvent such as a buffer at the time of use may be used. Further, the holding may be carried on the inner wall surface of the recess, or may be simply housed in the inside by closing the opening with a sheet that is removed during use, etc. It may be carried on a closing sheet.

In principle, the reagents 11 to be held in the recesses 10 are of different types depending on the detection target, and a variety of detection targets are discriminated by applying the same test object to each recess. It is. For example, in the case of detecting various nucleic acids in the same test object, a primer is matched with the corresponding nucleic acid in each recess 10 according to the nucleic acid to be detected and held as a reagent 11 to be tested. After applying the product, nucleic acid is amplified and reacted with the coloring agent in the reagent 11 to determine the presence or absence of various nucleic acids from the presence or absence of coloration or discoloration. If there is some relationship between the degree of coloration or color change of the coloring agent and the amount of the detection target, such as linearity, the amount and concentration of the detection target can be measured. is there. However, if it is necessary to confirm the coloration or discoloration of the plurality of recesses 10 with respect to the same detection target due to various circumstances, the same reagent is used for each of the plurality of recesses 10. May be held.

The imaging means 2 may be anything as long as it has a function capable of imaging at least the whole of the plurality of recesses 10 of the inspection apparatus 1 at a time. For example, a camera such as a digital camera may be used, and a camera or a CCD camera incorporated in various portable terminals such as a mobile phone, a smart phone, or a tablet computer may be used. Moreover, in order to obtain clear imaging data stably, what has an illumination means is also preferable. Illumination means includes incandescent bulbs, fluorescent lamps, LED lighting, and the like. By appropriately providing these means, it is possible to acquire imaging data without problems both indoors at 5000 lux or less and outdoors at 100,000 lux or less. When a fluorescent labeling reagent is used as a coloring agent, a mercury lamp, a xenon lamp, an ultraviolet irradiation device, a laser irradiation device, or the like that emits excitation light can be used, and the imaging means is a fluorescent filter for capturing fluorescence. Or a mirror.

The computer 3 is composed of a single computer or a plurality of computers. As shown in FIG. 4, a conventionally used computer device including a storage unit 31 and a communication control unit 32 around a processing device 30 can be used. The processing device 30 is mainly configured by a CPU such as a microprocessor, and has a storage unit including a RAM and a ROM (not shown), and stores programs and processing data for defining procedures of various processing operations.

Functionally, the processing device 30 includes an imaging data storage processing unit 30a that stores imaging data including a coloration or discoloration pattern received from a client terminal through the communication control unit 32 in the imaging data storage unit 31a, and an imaging unit 2. A determination processing unit 30b as a determination unit that determines the presence or absence of each detection target by comparing the coloration or discoloration pattern 18 of the whole of the plurality of recesses 10 to be imaged with the arrangement of the recesses stored in the storage unit 31; A determination result creation processing unit 30c that creates a document for returning the determination result to the client terminal; and a determination result transmission processing unit 30d that transmits the determination result document data to the client terminal 4 through the communication control unit 32; These functions are realized by the above program.

The storage unit 31 includes a hard disk or the like inside or outside the computer 3, and includes an imaging data storage unit 31 a that stores imaging data including a coloring or discoloration pattern received from the client terminal 4, and the plurality of coloring or discoloration of the inspection apparatus 1. An array information storage unit 31b that stores the array information of the pattern 18, a detection target storage unit 31c that stores a detection target in each concave portion of the plurality of coloration or discoloration patterns 18, and a prediction according to the type of the detection target that exists A determination result document storage unit 31d for storing the determination result document to be processed. Such storage means 31 includes, of course, a case of storing in a temporary storage area in addition to the hard disk and the like.

More specifically, the determination processing unit 30b compares the coloration or discoloration pattern 18 of the imaging data with the arrangement information in the arrangement information storage unit 31b, and extracts the depression or depressions that are colored or discolored. The processing unit 33a and a detection target specifying processing unit 33b that specifies the detection target of the extracted colored or discolored concave portion based on information in the detection target storage unit 31c.

Since the opening shape of the concave portion that has not been colored or discolored appears in the imaging data, the concave portion extraction processing unit 33a first identifies all the concave portions on the imaging data in comparison with the arrangement information, and further presents the concave shape. This can be done by identifying a color or discolored recess. These identifications can be performed using a known image analysis method.

The colored or discolored image may be a color image or a gray scale image. Further, when processing in gray scale, a gray scale image may be captured at the time of imaging, or a process of converting to a gray scale after imaging with a color image may be performed. However, in the case of using a reagent that colors or changes color in each concave portion, it is desirable to determine the presence or absence of a detection target from a color image, but it may be determined from a gray scale image if possible. The determination of the presence or absence of coloration or discoloration can be made based on, for example, whether the color density exceeds a certain threshold value. When measuring the amount or concentration of the detection target, prepare one to three rows of concave portions for preparing a calibration curve as in the ELISA method, and compare the degree of coloration or discoloration with the object to be inspected. A method of calculating the concentration of A may be used. Further, as shown in FIG. 5A, a known pattern method such as printing or sticking is used on the upper surface of the inspection apparatus 1, as shown in FIG. A method is also suitable in which the density is calculated by comparing with the degree of coloration or discoloration of the object A to be inspected. In particular, when measuring the concentration of nucleic acid in the test object A, the concentration of nucleic acid can be measured with high accuracy by using a triarylmethane dye such as a leuco dye as a coloring agent for nucleic acid detection. Is possible. In such a quantitative measurement system, from the viewpoint of ensuring the reproducibility and accuracy of the reaction, as in the ELISA method described above, several rows of concave portions for preparing a calibration curve are prepared and reacted with the specimen A body, Generally, the concentration of the measurement target substance in the inspection object A is calculated using the obtained calibration curve data. However, since the leuco dye has the property that it binds almost in proportion to the amount of nucleic acid, it can be quantitatively measured with no problem in terms of reproducibility and accuracy by a simple method of comparison with the light and shade pattern 51. Is possible. In addition, a dye that is widely used in each technical field may be used instead of the leuco dye. In this case, if there is no problem in the reproducibility and accuracy of the reaction, quantitative measurement may be performed using the above-described gray pattern, but other than that, for example, each concave portion in the gray scale image It is good to calculate with the signal level of, or with the RGB vector of each recess in the color image.

Alternatively, a method may be used in which a plurality of reference coloration or discoloration patterns are prepared in advance and a pattern that matches or approximates this is extracted. The imaging data is taken from various directions, angles, and distances depending on the shooting posture, but one or more reference marks on the upper surface of the inspection apparatus 1 and a reference recess (for example, by positive control) are set. In addition, based on this, the rotation angle, inclination, and distance (size) may be corrected by known means.

The client terminal 4 is a mobile terminal 5 that includes a camera, a wireless communication control unit that transmits and receives information to and from the computer, and information display means. For example, a mobile phone, a smart phone, a PDA, a notebook personal computer or other mobile terminals can be used. Accordingly, the user can easily send the imaging data to the computer and obtain the determination result from various sites indoors and outdoors, from inside a car, an airplane, a train, or the like. Further, if there is appropriate analysis software in the terminal, the determination can be made only by the terminal. In addition, although it is preferable that the client terminal 4 (mobile terminal 5) is configured as a cloud system that does not store the determination result to be displayed, the present invention is not limited to this.

FIG. 5 is an example provided with temperature adjusting means 8 for accelerating the reaction between the test object A and the reagent 11 placed in the recess 10 of the inspection apparatus 1. By providing such a temperature control means 8, a step of amplification during detection as in the case where the detection target is a nucleic acid or a growth process as in the case of a microorganism is required. It is possible to deal with cases that are essential. In this example, an example is shown in which the temperature adjusting means 8 is prepared separately from the inspection apparatus 1, but the temperature adjustment means 8 may be integrated with the inspection apparatus 1. In particular, when the temperature control means 8 is used as a means for amplifying nucleic acid, an amplification method such as a LAMP method, an ICAN method, a PALSAR method, or a SmartAmp method that can use an isothermal nucleic acid amplification device can be applied. A simple and inexpensive temperature control device such as a slide warmer or a thermostatic bath can be used. Such temperature adjusting means 8 may be configured so that power can be supplied from the portable terminal 5 as shown in the figure, or other known power supply system can be connected, or the power supply system can be connected to the temperature adjusting means 8. Or may be configured so as to be incorporated. Moreover, it is also preferable to be configured to be connectable to a portable solar power generation device in consideration of power shortage outdoors or in the mobile terminal 5.

Hereinafter, the procedure of the inspection method will be described with reference to FIGS.

First, the same inspected object A is applied to the respective recesses 10 of the inspection apparatus 1 shown in FIG. 2A by a predetermined amount with the applying tool 60 as shown in FIG. 2B. Here, when the amount of the inspection object A to be applied does not need to be accurate, for example, as shown in FIG. It is also possible to apply all the concave portions together. In principle, different reagents 11 are held in each recess 10, and the object to be inspected reacts with the reagent 11 in each recess 10 by applying, so that a detection target corresponding to each reagent 11 exists. In this case, the concave portion 10 having the reagent 11 is colored or discolored, and the color or discolored pattern 18 of the plurality of concave portions 10 is formed on the upper surface of the inspection apparatus 1.

In this state, as shown in FIG. 2 (c), a recess is formed from above the inspection apparatus 1 so that all the recesses 10 can be accommodated by the camera (imaging means 2) of the mobile terminal 5 as the client terminal 4 based on a user operation. The entire 10 coloration or discoloration pattern 18 is imaged. Next, as shown in FIG. 3A, the mobile terminal 5 transmits imaging data 6 composed of a coloration or a discoloration pattern to the computer 3 based on a user operation.

Then, the computer 3 compares the coloration or discoloration pattern of the imaging data 6 received from the portable terminal 5 with the arrangement of the concave portions of the storage means 31 to determine the presence or absence of each detection target, as shown in FIG. Thus, the determination result document data 7 is transmitted to the portable terminal 5, and the portable terminal 5 displays the determination result document 19 received from the computer 3 on the information display means 50.

The processing procedure by the computer 3 from the reception of the imaging data to the transmission of the determination result document will be described in more detail based on FIG. 4 and FIG.

When imaging data is transmitted from the portable terminal 5 to the computer 3 (S101), the computer 3 first stores the imaging data received by the imaging data storage processing unit 30a in the imaging data storage unit 31a (S102). Next, the concave portion extraction processing unit 33a compares the coloration or discoloration pattern of the imaging data extracted from the imaging data storage unit 31a with the arrangement information of the arrangement information storage unit 31b, and is colored or discolored. Are detected (S103), and the detection target specifying processing unit 33b specifies the detection target of the extracted colored or discolored recess based on the information in the detection target storage unit 31c (S104).

Next, the determination result creation processing unit 30c creates a document for returning the determination result to the portable terminal 5 (S105), and the determination result transmission processing unit 30d sends the determination result document data to the portable terminal through the communication control unit 32. 5 (S106). The mobile terminal 5 that has received the determination result document data displays the received determination result document on the information display means 50 (S107). According to the present invention, it is possible to provide an inspection system and an inspection method capable of analyzing an object to be inspected on site and easily and quickly grasping an analysis result accurately without specialized knowledge.

It should be noted that information such as determination results created by the computer 3 is preferably stored in the computer 3 or another computer and used for information processing. For example, when the client terminal 4 (mobile terminal 5) has a GPS function (a function for acquiring its own position information) or a function for acquiring the position information of the wireless communication base station, and transmits the imaging data 6 to the computer 3 If this position information is also transmitted, the computer 3 can manage the determination result in association with the position information. Similarly, when the imaging data 6 is transmitted from the client terminal 4 to the computer 3, the personal information of the subject from which the inspection object A is derived is input and transmitted at any time in the world or in Japan. It is also possible to grasp in real time what kind of person is causing what kind of disease and how much (number of people) in which region and place. In addition, for the purpose of controlling hospital-acquired infections at medical institutions, a hospital-acquired infection map is created, and it is useful to formulate a plan for what kind of room to respond to and contain infection. You can also. The same can be used for grasping not only diseases but also the spread of environmental pollution. The recent pandemic of influenza has shown how important this type of information was, and when this information is accumulated, analyzed, and utilized, graphs showing the number of diseases on a map (by country or region) , Age, sex, etc.) are also preferably provided. By analyzing such information, it is possible to quickly formulate and execute preventive measures such as appropriate diseases and environmental pollution. It is preferable that such information can be viewed on the client terminal.

Hereinafter, a modified example constituted by a portable computer alone will be described. In this case, as shown in FIG. 7, the portable computer 3A is provided with an imaging unit, a storage unit, a determination unit, a display unit, and the like, and the process from imaging to display of the determination result is performed. Specifically, as shown in FIG. 7, a storage unit 31, an imaging unit 2, and an information display unit 50 are provided around a processing device 30, and the processing device 30 is mainly configured by a CPU such as a microprocessor. In addition, a program and processing data that have a storage unit including a RAM and a ROM (not shown) and that define procedures for various processing operations are stored. As such a computer 3A, for example, a mobile phone, a smart phone, a PDA, a notebook personal computer, and other mobile terminals can be used as in the mobile terminal 5 in the representative embodiment described above. In this case, these processing devices 30 include test target information, subject information (name, sex, age, address, telephone number, e-mail address, medical record number, medical history, blood type, genetic information, etc.) and test position information. Etc. may be input as appropriate. Further, a password management function may be provided as a security function.

Functionally, the processing device 30 has an imaging data storage processing unit 30a that stores imaging data of the coloration or discoloration pattern 18 of the whole of the plurality of recesses 10 imaged by the imaging unit 2 in the imaging data storage unit 31a; A determination processing unit 30b, a determination result generation processing unit 30c that generates a document to be displayed on the information display unit 50, and a determination result display processing unit 30e that performs a process of displaying the determination result document data on the information display unit 50 These functions are realized by the above program. The determination processing unit 30b, the determination result creation processing unit 30c, and the storage unit 31 are the same as the configuration of the computer 3 in the representative embodiment described above.

In this modification, the processing procedure in the computer 3A from imaging to display of the determination result is as shown in FIG. That is, when the coloration or discoloration pattern 18 of the entire plurality of recesses 10 is imaged by the imaging means 2 of the computer 3A (S201), the imaging data acquired by the imaging data storage processing unit 30a is stored in the imaging data storage unit 31a. (S202). Next, the concave portion extraction processing unit 33a compares the coloration or discoloration pattern of the imaging data extracted from the imaging data storage unit 31a with the arrangement information of the arrangement information storage unit 31b, and is colored or discolored. Are detected (S203), and the detection target specifying processing unit 33b specifies the detection target of the extracted colored or discolored recess based on the information in the detection target storage unit 31c (S204). Next, the determination result creation processing unit 30c creates a determination result document (S205), and the determination result display processing unit 30e displays the determination result document on the information display unit 50 (S206).

The embodiments of the present invention have been described above. However, the present invention is not limited to these examples, and it is needless to say that the present invention can be implemented in various forms without departing from the gist of the present invention.

Hereinafter, the embodiments of the present invention will be described more specifically based on examples, but the present invention is not limited to these.

(Example 1)
[Pathogen detection test]
1) Imaging analysis device: A smart phone (ARROWS NX F-06E) was prepared, which can input an inspection device ID and incorporated a coloring position analysis function of a captured image.
2) Extracted DNA: The supernatant obtained by heat-treating 100 μL of each type of ae to H. influenzae culture solution at 100 ° C. for 10 minutes and centrifuging at 10,000 rpm for 3 minutes was used as an extracted DNA solution.
3) Inspection chip: A polypropylene inspection chip (see FIG. 1 (c)) in which holes of 2 mm in diameter and 3 mm in depth, which were produced by cutting, were arranged in 5 × 5, was prepared as an inspection apparatus. Next, after adding 0.36 μL of nucleic acid detection reagent (0.1% gentian violet B, 2.1% sodium sulfite, 1.5% β cyclodextrin) to all holes, dry with hot air at 50 ° C. By performing, the nucleic acid detection reagent was held in each hole. The hole numbers were set from 1 to 5 from left to right in the uppermost stage and from 6 to 10 from left to right in the lower stage.
3-1) Imaging analysis using test chip (LAMP reaction version): In test chip as shown in FIG. 1 (c), H. influenzae type a (sequence numbers 1 to 6) Hole Nos. 6 to 10 have Haemophilus influenzae type b (SEQ ID Nos. 7 to 11), Hole Nos. 11 to 15 have Haemophilus influenzae type c (SEQ ID Nos. 12 to 15), and Nos. 16 to 20 have Haemophilus influenzae type d ( A primer mix of Haemophilus influenzae type e (SEQ ID NOs: 20 to 23) was added to SEQ ID NOs: 16 to 19) and hole numbers 21 to 25. Each primer mix has a final concentration of SEQ ID NO: 1, 2, 7, 8, 12, 13, 16, 17, 20, 21 of 1.6 μM, SEQ ID NOs: 3, 4, 9, 10, 14, 15, 18, Nos. 19, 22, and 23 were mixed in advance so that 0.2 μM, LF, and SEQ ID NOs: 5, 6, and 11 were 0.8 μM. The template DNAs are H. influenzae type A in

hole numbers

1, 3, 5; H. influenzae b type in

hole numbers

7 and 9; H. influenzae c type in

hole numbers

11, 13 and 15; H. influenzae d in

hole numbers

17 and 19; 2 μL of Haemophilus influenzae type e was applied to the type and hole numbers 21, 23 and 25. 2 μL of sterilized water was applied to the holes to which no extracted DNA was added. Next, from a commercially available LAMP reaction kit (Loopamp DNA amplification kit, manufactured by Eiken Chemical Co., Ltd.), 4.5 μL of 2 × Reaction Mix and 0.5 μL of Bst DNA Polymerase were added to all the holes.
Thereafter, the test chip was covered with a transparent plastic lid, and then the test chip was placed on a glass plate (manufactured by Blast Co., Ltd.) that can be heated and kept warm, and kept at 65 ° C. for 60 minutes for gene amplification. Thereafter, the reaction was stopped by heating at 80 ° C. for 2 minutes, and the reaction was returned to room temperature. As a result, 13 sites (

hole numbers

1, 3, 5, 7, 9, 11) to which the detection primer and the extraction DNA as a template were added were added. , 13, 15, 17, 19, 21, 23, 25), the reaction solution was colored blue.
When the inspection device ID is input to the imaging analyzer and the image of the chip colored amplification product is taken indoors (under 500 lux),

hole numbers

1, 3, and 5 are positive for H. influenzae type a,

hole number

7 and 9 are H. influenzae type b positive,

hole numbers

11, 13, and 15 are H. influenzae type c positive,

hole numbers

17 and 19 are H. influenzae type d positive, and hole numbers 21, 23, and 25 are H. influenzae type e positive. The image was displayed on the screen, and it was confirmed that the coloration due to amplification was recognized by the image pickup in accordance with the amplification result.

(Example 2)
1) Imaging analysis device: A smartphone (ARROWS NX F-06E) that can input the region (world map), gender (male or female), age, and inspection device ID and incorporates the coloring position analysis function of the captured image was prepared. .
2) Extracted DNA: After adding 50 μL of 50 mM sodium hydroxide aqueous solution to 5 μL of whole blood, heat treatment was performed at 100 ° C. for 10 minutes, and centrifugation was performed at 10,000 rpm for 3 minutes to obtain an extracted DNA solution. .
2-1) Imaging analysis using a test chip (SmartAmp reaction version): 2 μL of DNA solution extracted from whole blood in all holes of a test chip made of polypropylene charged with a nucleic acid detection reagent as in 3) of Example 1 Applied. Next, ALDH2 (wild type) detection primer set (SEQ ID NOs: 40, 41, 42, 44, 45) is added to

hole numbers

1, 3, 5 and 11, 13, 15 and

hole numbers

7, 9, 17, 19 are used. In addition, 2 μL each of ALDH2 (mutant) detection primer set (SEQ ID NOs: 40, 41, 43, 44, 45) was added. 2 μL of sterilized water was added to the holes where no primer was added.
Next, 4.5 μL of 2 × Reaction Mix and 0.5 μL of Aac DNA polymerase were added to all the holes from a commercially available SmartAmp reaction kit (manufactured by DNAFORM). Thereafter, a transparent plastic lid was placed on the test chip, and then the test chip was placed on a glass plate that can be heated and kept warm, and kept at 65 ° C. for 60 minutes for gene amplification. Then, when the reaction was stopped by returning to room temperature by heat treatment at 80 ° C. for 2 minutes, 6 positions of

hole numbers

1, 3, 5, 11, 13, 15 to which the ALDH2 (wild type) primer set was added were added. The reaction solution was colored blue.
When sex (male) / age (40 years old) / inspection device ID is input to the imaging analyzer and the image of the chip colored amplification product is taken indoors (under 500 lux), ALDH2 (wild type) is positive , ALDH2 (mutant) negative was displayed on the screen, which was consistent with the amplification result.

(Example 3)
Imaging analysis was performed outdoors (under 10,000 lux conditions) using the plate after amplification and coloring in Example 1. As a result, it was found that an analysis result that coincides with the amplification result is displayed as in the case of indoors, and that an imaging analysis can be performed accurately even outdoors.

Example 4
[Quantitative evaluation test 1]
Imaging analysis using a test chip: The H. influenzae type b detection primer set used in Example 1 was prepared in the hole numbers 1 to 10 of the same test chip as used in Example 1, and further, at the end of the test chip, A sticker with a blue gradient (white → blue) was applied. In the image analysis, the darkest blue color is set to 1 and the white color is set to 0.
The extracted DNA solution used in 2) of Example 1 was not diluted in

hole numbers

1 and 5, 10 times in

hole numbers

2 and 6, 100 times in

hole numbers

3 and 7, and in

hole numbers

4 and 8. Was diluted 1000 times and 2 μL was applied. In addition, 2 μL of sterilized water was applied to the

hole numbers

5 and 10. When gene amplification was carried out in the same reaction system as in Example 1, the blue coloration became lighter in the order of

hole numbers

1, 2, 3 and 4 (

hole numbers

5 and 10 to which no extracted DNA was added are shown in FIGS. colorless). This is imaged at the same time as the above-mentioned seal, and the contrast is automatically converted into a numerical value by using a smartphone. The hole numbers 1 and 6 are 0.6, the

hole numbers

2 and 7 are 0.4, and the hole number. 3 and 8 were displayed as 0.2, and

hole numbers

4 and 9 were displayed as 0.1. As a result, it was possible to analyze and display the color shading.

(Example 5)
[Quantitative evaluation test 2]
Imaging analysis using test chip: The same operation as in Example 4 was performed, except that the test chip charged with the ALDH2 detection primer set used in Example 2 was used.
The extracted DNA solution used in 2) of Example 2 was not diluted in

hole numbers

1 and 5, 10 times in

hole numbers

2 and 6, 100 times in

hole numbers

3 and 7, and in

hole numbers

5 and 10. When gene amplification was carried out in the same reaction system as in Example 2, the blue coloration became lighter in the order of

hole numbers

1, 2, 3 and 4 (

hole numbers

5 and 10 to which no extracted DNA was added are shown in FIG. colorless). This is imaged at the same time as the above-mentioned seal, and the contrast is compared with the seal and the shading is automatically converted into numerical values using a smart phone. Hole numbers 1 and 6 are 0.6,

hole numbers

2 and 7 are 0.4,

hole numbers

3 and 8 were displayed as 0.2, and

hole numbers

(Example 6)
[Quantitative evaluation test 3]
Imaging analysis using a test chip: A blue gradient (white → blue) is attached to the end of a test chip in which the primer sets for amplification of the multi-cloning site of pUC18 (SEQ ID NOs: 46 and 47) are prepared in hole numbers 1 to 10. A sticker was attached. In the image analysis, the darkest blue color is set to 1 and the white color is set to 0.
As a template DNA, pUC18 aqueous solution was diluted in holes 1 and 5 without dilution, 10 times in

holes

2 and 6, 100 times in

holes

3 and 7, and 1000 times in

holes

4 and 8. 2 μL was applied. In addition, 2 μL of sterilized water was applied to the

hole numbers

5 and 10. Next, 0.9 μL of 10 × buffer Mix, 1.5 μL of dNTP, 0.1 Taq DNA polymerase, and 4.5 μL of sterilized water were added from a commercially available PCR kit (manufactured by Takara Bio Inc.). After covering with a transparent plastic cover, the chip is placed on a glass plate that can be heated and kept warm, and a gene amplification is performed by performing 30 cycles of 95 ° C for 1 minute, 60 ° C for 30 seconds, and 72 ° C for 30 seconds. It was. Thereafter, when the reaction solution was returned to room temperature, the blue color became lighter in the order of

hole numbers

1, 2, 3, and 4 (

hole numbers

5 and 10 to which no template DNA was added were colorless). This is imaged at the same time as the above-mentioned seal, and the contrast is automatically converted into a numerical value by using a smartphone. The hole numbers 1 and 6 are 0.6, the

hole numbers

2 and 7 are 0.4, and the hole number. 3 and 8 were displayed as 0.2, and

hole numbers

(Example 7)
[Biological sample evaluation test]
A specimen (nasopharyngeal secretion) was collected from the posterior nasal cavity using a sterilized rayon swab, and 100 μL of an extracted DNA solution was obtained using a commercially available DNA extraction kit (QIAamp DNA Micro Kit, QIAGEN).
6-1) Imaging analysis using test chip: In the same manner as in 3) of Example 1, 2 μL of the extracted DNA solution of nasopharyngeal secretion was applied to all holes of the plastic charged with the nucleic acid detection reagent. Haemophilus influenza detection primer set (Sequence numbers 35-39) in hole numbers 1-4, influenza a type detection primer set in hole numbers 6-9, Haemophilus influenzae type b, hole number 16 in hole numbers 11-14 A detection primer set of Haemophilus influenzae type c was added to -19 and influenza d type was added to hole numbers 21-24. Sterile water was added to the hole numbers where no primer was added.
Next, from a commercially available LAMP reaction kit (Loopamp DNA amplification kit, manufactured by Eiken Chemical Co., Ltd.), 4.5 μL of 2 × Reaction Mix and 0.5 μL of Bst DNA polymerase were added to all the holes. After covering with a transparent plastic lid, the chip was placed on a glass plate that can be kept warm and kept at 65 ° C. for 60 minutes to perform gene amplification. Thereafter, the reaction was stopped by heat treatment at 80 ° C. for 2 minutes, and the reaction was returned to room temperature. As a result, the four reaction solutions in the hole numbers 1 to 4 to which the Haemophilus influenza detection primer set of the bacterium was added were colored blue.
When the sex (male), age (40 years), plate ID (Haemophilus influenza test chip) was input into the imaging analyzer, and the chip image colored with the amplification product was taken indoors (under 500 lux), Haemophilus A positive influenza screen was displayed, which was consistent with the amplification results.

(Example 8)
In the same manner as in Example 1, a nucleic acid detection reagent was charged in all 96 holes of a 96-well plate for PCR (manufactured by Nippon Genetics). Hole numbers 1 to 11 are H. influenzae type a, hole numbers 13 to 23 are H. influenzae type b, hole numbers 25 to 35 are H. influenzae type c, hole numbers 37 to 47 are H. influenzae type d, hole numbers 49-59 is H. influenzae type E, hole numbers 61-71 are H. influenzae type f (SEQ ID NO: 24-29), hole numbers 73-83 are Pneumococcus (SEQ ID NO: 30-34), hole numbers 85-95 Was added with a Haemophilus influenza primer mix. Each primer mix used previously was mixed in the same manner as in Examples 1 and 2. 2 μL of template DNA was applied to all odd-numbered hole numbers. In addition, 2 μL of sterilized water was applied to the holes where the primer mix was not added. Next, from a commercially available LAMP reaction kit (Loopamp DNA amplification kit, manufactured by Eiken Chemical Co., Ltd.), 4.5 μL of 2 × Reaction Mix and 0.5 μL of Bst DNA Polymerase were added to all the holes. After covering with a transparent plastic lid, the chip was placed on a glass plate (manufactured by Blast Co., Ltd.) that can be kept warm, and kept at 65 ° C. for 60 minutes for gene amplification. Thereafter, the reaction was stopped by heating at 80 ° C. for 2 minutes, and the temperature was returned to room temperature. As a result, the reaction solution in 48 holes to which the detection primer and the extracted DNA as a template were added was colored blue.
When the image of the 96-well plate colored with the amplification product was taken indoors (under 500 lux) with an imaging analyzer,

hole numbers

1, 3, 5, 7, 9, and 11 were positive for H. influenzae type a ,

Hole numbers

13, 15, 17, 19, 21, and 23 are positive for H. influenzae type b, hole numbers 25, 27, 29, 31, 33, and 35 are positive for H. influenzae type c, and hole numbers 37, 39, 41, and 43 45, 47 are H. influenzae d-type positive, hole numbers 49, 51, 53, 55, 57, 59 are H. influenzae e-type positive, and hole numbers 61, 63, 65, 67, 69, 71 are H. influenzae f-type positive , Hole numbers 73, 75, 77, 79, 81, 83 were indicated as positive for Pneumococcus, and hole numbers 85, 87, 89, 91, 93, 95 were indicated as positive for Haemophilus influenza. In agreement with the amplification result, it was confirmed that coloring due to amplification was recognized by imaging.

DESCRIPTION OF SYMBOLS 1 Inspection apparatus 2 Imaging means 3 Computer 4 Client terminal 5 Portable terminal 6 Imaging data 7 Judgment result document data 8 Temperature control means 10 Recess 11 Reagent 12 Plate body 12a Upper surface 13 Tube 14 Tube rack 15 Inspection chip 16 Reference mark 17 Bar code 18 Coloration or discoloration pattern 19 Determination result document 30 Processing device 30a Imaging data storage processing unit 30b Determination processing unit 30c Determination result creation processing unit 30d Determination result transmission processing unit 30e Determination result display processing unit 31 Storage means 31a Imaging data storage unit 31b Sequence information storage unit 31c Detection target storage unit 31d Determination result document storage unit 32 Communication control unit 33a Concave extraction processing unit 33b Detection target specifying processing unit 50 Information display means 60 Appliance for applying A A test object

Claims

An inspection system for analyzing and investigating the presence or absence of multiple types of objects to be inspected,
In addition to a plurality of recesses for applying the object to be inspected, a reagent containing a coloring agent that is a reagent corresponding to the detection target and that is colored or discolored due to the presence of the detection target is previously provided in each recess. A holding inspection device;
Imaging means for imaging the plurality of recesses of the inspection device;
Storage means for storing the array of the plurality of recesses and the detection target of each recess of the inspection apparatus, and a coloration or discoloration pattern of the whole of the plurality of recesses imaged by the imaging means, and an array of the recesses of the storage means A computer having a determination means for determining the presence or absence of each detection target by comparing
An inspection system characterized by comprising:
2. The inspection system according to claim 1, wherein the reagent contains a reaction promoting substance that amplifies or proliferates the detection target.
The inspection system according to claim 1 or 2, wherein the detection target is a nucleic acid.
4. The inspection system according to claim 1, wherein the coloring agent is a leuco dye.
The inspection system according to any one of claims 1 to 4, further comprising temperature adjusting means for promoting a reaction between the inspection object applied to the recess and the reagent.
The imaging means includes a camera that images the plurality of recesses, a communication control means that transmits and receives information to and from the computer, and a client terminal that includes information display means.
6. The imaging data comprising the coloration or discoloration pattern imaged by the camera is transmitted from the client terminal to the computer, and the determination result received from the computer is displayed on the information display means of the client terminal. The inspection system according to any one of the above.
The inspection system according to claim 6, wherein the client terminal is a portable terminal having a wireless communication control unit as the communication control means, and is connected to the computer via a wireless communication network.
An inspection method for analyzing and investigating the presence or absence of multiple types of objects to be inspected,
In addition to a plurality of recesses for applying the object to be inspected, a reagent containing a coloring agent that is a reagent corresponding to the detection target and that is colored or discolored due to the presence of the detection target is previously provided in each recess. An inspection device that is held; an imaging unit that images the plurality of recesses of the inspection device; a storage unit that stores an array of the plurality of recesses of the inspection device and a detection target of each recess; and the imaging unit A computer provided with a determination means for determining the presence or absence of a detection target by comparing the coloration or discoloration pattern of the whole of the plurality of recesses to be imaged with the arrangement of recesses of the storage means
Applying a test object to a plurality of recesses of the inspection device; and
A procedure for imaging the coloration or discoloration pattern of the entire plurality of recesses by the imaging means;
A procedure for the computer to determine the presence or absence of each detection target by comparing the coloration or discoloration pattern imaged by the imaging means and the arrangement of the concave portions of the storage means;
Inspection method consisting of.
The imaging means includes a camera that images the plurality of recesses, a communication control means that transmits and receives information to and from the computer, and a client terminal that includes information display means.
Applying a test object to a plurality of recesses of the inspection device; and
A procedure in which the client terminal images a coloration or discoloration pattern of the whole of the plurality of recesses by the camera;
A procedure in which the client terminal transmits imaging data including the coloration or discoloration pattern captured by the camera to the computer;
The computer determines the presence or absence of each detection target by comparing the coloration or discoloration pattern of the imaging data received from the client terminal with the array of recesses in the storage means;
A procedure in which the computer transmits a determination result to the client terminal;
A procedure in which the client terminal displays the determination result received from the computer on the information display means;
The inspection method according to claim 8, further comprising: