JP2014232064A - Bacteria detection method and detection device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify overall handling by eliminating the mixing in use at the outside of a system of a bacteria extract in the bacteria detection using a device and accompanied with a biofilm, while maintaining goodness of the detection sensitivity.SOLUTION: A bacteria detection method and a device for performing the detection method are characterized as follows: a detection unit for representing the presence of bacteria to be detected through a signal of antigen-antibody reaction is provided on a solid phase of the device; a carrier unit for carrying a specific component of producing a component decomposing the biofilm of bacteria cells in reaction to dissolved components of a dilute solution in a sample is provided separately from the detection unit; the bacteria biofilm in the sample is decomposed by that the component for decomposing the biofilm is produced in the carrier unit by contact in the carrier unit of the sample in which an analyte is extracted by the dilute solution; and the biofilm detects the bacteria to be detected by detecting the signal of the antigen-antibody reaction due to the decomposed bacteria component.

Description

  The present invention relates to a means for detecting microorganisms, and more specifically relates to a method capable of efficiently detecting a bacterium while dissolving a biofilm associated with the bacterium, and a detection instrument using the method. is there.

  A biofilm is a structure formed by bacteria attached to a substrate secreting an extracellular polysaccharide (EPS). EPS plays a role of a barrier and transportation route, and bacteria are protected from environmental changes and chemical substances.

  On the other hand, the detection of bacteria is performed for various reasons in various types of bacteria, and the necessity of such inspection in the modern society is very large.

  As a typical means for detecting a bacterium, a test using an antigen-antibody reaction as a detection signal using an antibody specific to a characteristic component or part of a bacterium to be detected is used. The biofilm is an obstacle to the detection method using such antibodies against bacteria.

  In other words, characteristic components of bacteria are often present in the cell membrane, and if the bacterial cell membrane is covered with a biofilm, it will be difficult to accurately detect the target bacteria. Is recognized.

  One example is Streptococcus ryogenes. Streptococcus pyogenes is a kind of eubacteria belonging to the genus Streptococcus. When it is cultured on a blood agar medium, it exhibits β-hemolytic properties and belongs to the group A in the Lancefield antigen classification used for differentiation of streptococci. Because of this property, Streptococcus pyogenes is also called group A β-hemolytic streptococci. Furthermore, it may be abbreviated as group A streptococci, or simply streptococci, and further GAS (Group A Streptococci).

  Streptococcus pyogenes is a kind of resident bacteria that also live in the pharynx, digestive tract, and epidermis of healthy humans. It causes various diseases such as sexually transmitted diseases or immune diseases that occur as a kind of complication after infection. In some cases, it can cause a fast-acting lethal disease called fulminant streptococcal infection.

  1 and 2 show an example of the contents of a conventional detection kit by Streptococcus pyogenes immunochromatography (Non-Patent Document 1). FIG. 1 shows the configuration of the kit, and FIG. 2 shows the detection procedure for Streptococcus pyogenes using the kit.

  A conventional detection kit 10 for Streptococcus pyogenes includes a specimen extract-sealed container 1 (a set of a container 11 in which an extract A is enclosed and a container 12 in which an extract B is enclosed), a reaction tube 2, a cotton swab 3, and It is comprised by the solid phase 4 for a test.

  Extracts A and B are different from each other and are solutions of acetic acid or sodium nitrite. By mixing both solutions, nitric acid is generated, and the biofilm of Streptococcus pyogenes can be dissolved with this nitric acid. Extracts A and B must be mixed at the time of use.

  FIG. 2 (1) shows the on-use mixing of the above extract. Extract A (113) is dropped from the dripping port 111 of the container 11 into the opening 21 of the reaction tube 2, and extract B (114) is dropped from the dripping port 112 of the container 12. Is mixed into the mixed extract 115 to generate nitric acid in the liquid as described above.

  In FIG. 2 (2), the head 31 of the swab 3 is rubbed against the subject's palate to attach the specimen, and the head 31 of the swab 3 is mixed and extracted into the mixed extract 115 of the reaction tube 2. The specimen is extracted and dissolved in the liquid 115, and becomes a specimen extract 116 in which the bacterial biofilm to be detected is decomposed over time.

  In FIG. 2 (3), the solid phase 4 is provided with a detection unit 43 on which an antibody against Streptococcus pyogenes that generates a signal when an antigen-antibody reaction occurs is immobilized. With the handling portion 42 for the operator to touch, the opposite end 41 is immersed in the sample extract 116 and the solution 116 and the solid phase 4 are brought into contact with each other. Starts moving upward (in the direction of arrow a).

  When the specimen extract 116 reaches the detection unit 43 on the solid phase 4, when Streptococcus pyogenes exists in the specimen, the antibody against Streptococcus pyogenes carried on the detection unit 43 decomposes the biofilm. The S. pyogenes in the specimen can be detected by binding to Streptococcus pyogenes whose characteristic components are exposed by antigen-antibody reaction and confirming the binding signal by visual observation or the like.

  As another prior art, a technique is disclosed in which a solid phase substantially the same as the above-described solid phase 4 is fixedly arranged in a case, and the above reaction step is performed in the case (Non-patent Document 2). ).

Package insert of "Rabbit Tester (registered trademark) Strep A" for detecting group A β-hemolytic streptococcal antigens in throat swabs (Sekisui Medical Co., Ltd.) Package insert of "Elnas (registered trademark) Strep A" reagent for detecting group A β-hemolytic streptococcal antigens in pharyngeal specimens (TEFP Corporation)

  In the above-described prior art (Non-Patent Documents 1 and 2), it cannot be denied that there is a complicated aspect in handling in the medical field. That is, in the operation procedure in the prior art shown in FIG. 2, it is necessary to mix the extract A and B at the time of use as shown in (1), and it is necessary to leave this still for about 1 minute. At medical sites, doctors or nurses perform a series of operations from sample collection to confirmation of the presence / absence of a reaction. If you have to stand still and wait for the completion of the nitric acid formation reaction, a considerable sense of burden will occur if you accumulate them. In addition, there is no denying that it is easy to induce operational errors.

  These are problems to be solved by the present invention.

  As a result of intensive studies, the present inventor has found that the above-described problems can be solved by providing detection means having the following contents.

  That is, the present invention decomposes a biofilm of a bacterium to be detected on a solid phase, and detects the bacterium by a signal indicating an antigen-antibody reaction on the solid phase with respect to the bacterium component after the biofilm decomposition. In this method, on the solid phase, a detection unit is provided for revealing the presence of bacteria to be detected via a signal of an antigen-antibody reaction, and the dilution in the sample is performed separately from the detection unit. A sample (specimen extraction diluted solution) in which a specific component that produces a component that decomposes a biofilm of bacterial cells in response to a dissolved component of the liquid is supported and the sample is extracted with a diluent A component that decomposes the biofilm is produced in the supporting unit by contact in the supporting unit, the bacterial biofilm in the sample is decomposed, and the bacterial component in which the biofilm is decomposed is decomposed. Bacterial detection method (hereinafter also referred to as the detection method of the present invention), characterized in that a detection target bacterium is detected by detection of an antigen-antibody reaction signal generated by subsequent contact with the detection unit by movement in the solid phase. ).

  The “bacteria” that is the detection target of the detection method of the present invention is a bacterium that forms a biofilm, and is not particularly limited as long as it is. Examples include Streptococcus ryogenes (also known as group A β-hemolytic streptococci).

  The “solid phase” is not particularly limited as long as the sample extraction diluent (sample) that is a mobile phase can move, and examples thereof include non-woven fabric, woven fabric, paper, and porous material. Examples of the material include cotton, hemp, wool, silk, rayon, Bemberg, acetate, polyamide fiber, polyester, cellulose, nitrocellulose, glass fiber, polyethersulfone (PES) and the like. Further, the solid phase may be composed of a single element, but a plurality of materials may be combined as necessary. Rather, it is preferable to appropriately combine materials suitable for the elements and roles to be carried. The shape of the solid phase is not particularly limited, but the overall shape is usually a strip shape whose length direction is longer than the width direction. The thickness does not need to be the same as a whole, and may be changed as necessary. As will be described later, the material and shape of the solid phase are important factors in adjusting the movement time of the mobile phase.

  The component that decomposes the biofilm of bacteria is not particularly limited, and examples thereof include nitric acid and aqua regia. Nitric acid is preferable because it is necessary to produce the decomposition component step by step. In the present invention, the sample extraction diluent is brought into contact with the solid phase as a sample, and a component that decomposes the biofilm on the solid phase is generated in the supporting unit that supports a specific component that can be generated by reaction with the diluent. When the components dissolved in the diluent of the specimen extraction diluent (sample) come into contact with each other, a “biofilm decomposition component” such as nitric acid is generated.

For example, when the “biofilm degradation component” is nitric acid, the “diluent” and the “specific component” are any combination of the following (1) and (2), It is preferable that a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit.
(1) The diluted solution contains an organic acid, and the specific component is nitrite.
(2) The diluted solution contains nitrite, and the specific component is an organic acid.

  Examples of the nitrite include sodium nitrite and potassium nitrite. Examples of the organic acid include acetic acid and citric acid.

  The above-mentioned neutralizing component of nitric acid is supported on the solid phase after the biofilm is decomposed with nitric acid on the solid phase and before the mobile phase reaches the detection part, the pH of the mobile phase is set to near neutral. The purpose is to neutralize. And if it is a component in which it is possible, it will not specifically limit, For example, boric acid, phosphoric acid, a trishydroxymethylaminomethane (THAM: common name Tris) etc. are mentioned. By the action of the neutralizing component, the antigen-antibody reaction on the solid phase proceeds normally, and desired detection can be performed.

In the combination of the diluent and the specific component in any one of the above (1) and (2), suitable quantitative conditions for the diluent and the specific component are as shown in the following (A) and (B).
(A) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing is more than 0.04M and less than 0.14M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.06M or less or 0 Or (b) except when the organic acid concentration is 0.12M or more and the nitrite concentration is 0.06M or less).
(B) When the diluting solution contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluting solution exceeds 0.60 M but less than 1.4 M, and is supported in the solid phase. The organic acid concentration of the solution used for performing exceeds 0.06M and less than 0.16M (except when the nitrite concentration is 0.80M or less and the organic acid concentration is 0.14M or more) It is.

Further suitable quantitative conditions are as shown in the following (A ′) and (B ′).
(A ′) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing the above is more than 0.06M and less than 0.12M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.10M or more. Except in the case).
(B ′) When the diluent contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluent exceeds 0.60 M and less than 1.40 M, and is supported on the solid phase. The organic acid concentration of the solution used for carrying out is more than 0.06M and less than 0.14M (provided that (a) the nitrite concentration is 0.06M or less and the organic acid concentration is 0.08M or less, or Except for the case of 0.12M or more).

  Furthermore, the present invention is also an invention that provides a detection instrument for performing the above-described detection method of the present invention. The detection instrument is a method for decomposing a biofilm of a bacterium to be detected on a solid phase, and detecting the bacterium by a signal indicating an antigen-antibody reaction on the solid phase with respect to a component of the bacterium after the biofilm decomposition. On the solid phase, a detection unit for revealing the presence of bacteria to be detected via an antigen-antibody reaction signal is provided on the solid phase, and upstream of the detection unit, Bacterial detection instrument (hereinafter referred to as the present invention), characterized in that it is provided with a supporting part on which a specific component that generates a component that decomposes a biofilm of bacterial cells in response to a diluent in a sample is supported. It is also called the detection instrument of the invention).

  Further, the present invention provides a detection instrument of the present invention in which a solid phase is disposed and fixed in a case, and a sample and a diluent are mixed, and the mixed liquid is dropped toward the dropping window of the detection instrument. It is an invention providing a kit for detecting bacteria (hereinafter also referred to as a detection kit of the present invention) comprising a mixing and dropping device.

  In the present specification, claims, and drawings (hereinafter also referred to as the present specification and the like), the “upstream” in the solid phase is the carrier side, and the detection unit side is the “downstream” side. Suppose that

  In the present specification and the like, “solution” means “aqueous solution”, that is, a solution using water as a solvent, unless otherwise specified. In other words, the “aqueous solution” is a solution in which a water-soluble substance is dissolved in water, and includes various buffer solutions. Furthermore, the “aqueous solution” means an aqueous solution in which only the substance presented in the description is dissolved using water as a solvent. For example, “an aqueous solution of citric acid” means “an aqueous solution in which citric acid alone is dissolved in water”. An aqueous solution is a superordinate concept of an aqueous solution, and an aqueous solution includes an aqueous solution.

  In the detection method of the present invention and the detection instrument for performing this, and further in the detection kit, the production reaction of the component that degrades the bacterial biofilm is performed on the solid phase. There is no need for the user to mix the diluent and the specific component at the time of use, and it is possible to isolate the user from the biofilm decomposition component, which is excellent in safety. And it becomes unnecessary to ensure the waiting time of the user after performing the said mixing at the time of use, it is excellent in efficiency, and the burden and feeling of a burden on the field with respect to a detection operation can be remarkably reduced. That is, the present invention is an invention that provides a detection method, a detection instrument, and a detection kit for bacteria that produce biofilms that are extremely excellent in practicality.

It is drawing which showed the structure of the detection kit by the immunochromatography method of the conventional Streptococcus pyogenes. It is drawing which showed the detection procedure of Streptococcus pyogenes using the detection kit by the conventional immunochromatography method of Streptococcus pyogenes. It is an exploded view of the detection instrument of the present invention. It is a whole perspective view of the detection instrument of the present invention. It is the longitudinal cross-sectional view seen by cutting in the direction of arrow XX of the detection instrument of this invention. It is drawing which showed the structure of the detection kit of this invention. It is drawing which showed the mode at the time of the mixture extraction in the instrument for mixing dripping which is a component of the detection kit of this invention. It is drawing which showed the detection procedure of the detection target bacteria using the detection kit of this invention. In the detection kit of this invention, it is drawing which showed the mode of the dripping of the sample extraction dilution liquid (sample) from the mixing dripping instrument to a detection instrument.

  Hereinafter, an embodiment of the present invention will be disclosed based on the drawings. However, the scope of the present invention is based on the description in the claims, and is not limited to the description.

  3, 4 and 5 show an embodiment 50 of the detection instrument of the present invention, FIG. 3 is an exploded view of the detection instrument, FIG. 4 is an overall perspective view, and FIG. 1) and (2)) are longitudinal sectional views taken along the direction of arrow X shown in FIG.

  As shown in FIGS. 3, 4, and 5 (2), the detection instrument 50 of the present invention includes an upper exterior part 51, a lower exterior part 52, and a solid phase 53 for detection. The upper exterior part 51 and the lower exterior part 52 have a structure that can be fitted to each other. As shown in FIG. 5A, the case 50 'is formed by being fitted to each other. In addition, a structure 521 capable of mounting and fixing the solid phase 53 is provided inside the case 50 ′. The uneven portion indicated by 54 shown in FIG. 4 is a portion for preventing slippage when the detection instrument 50 is carried or operated.

  The upper exterior part 51 mainly shown in FIG. 3 is a component that constitutes the dropping window side of the exterior case of the detection instrument 50. That is, the upper exterior portion 51 is provided with an open dripping window 511 and a detection window 512 that ensures visibility when the window is viewed from the outside. The detection window 512 may be open or covered with a material such as transparent glass or transparent plastic as long as the internal visibility is ensured.

  The lower exterior part 52 mainly shown in FIG. 3 is a part of the exterior case that constitutes the floor surface side when the detection instrument 50 is put in use. The lower exterior portion 52 is provided with a rectangular region 521 ′ formed by a hook structure in which the solid phase 53 can be arranged and the movement of the arranged solid phase 53 in the planar direction can be fixed. The hook structure 521 ′ is a structure 521 in which the solid phase 53 shown in FIG. 5 (1) can be mounted and fixed by fitting the upper and lower exterior parts 51 and 52.

  The solid phase 53 shown in FIG. 3 or the like has a strip shape as a whole, and has a shape that fits into the rectangular region 521 ′. A carrying part 531 made of a thick absorbent fiber material is formed at a position corresponding to the position immediately below the dropping window 511 of the upper exterior part 51, and a neutralizing part made of a thick absorbent fiber material in contact with the downstream side thereof 532 is formed. Furthermore, a labeled antibody binding portion 533 is formed on the downstream substrate surface in contact with the neutralizing portion 532, in which the labeled antibody is immobilized on the solid phase substrate in a form that can move on the solid phase along with the movement of the aqueous solvent. Yes. A detection unit 532 is formed at a position corresponding to the position immediately below the detection window 512, and a control unit 535 is formed in the vicinity of the downstream portion thereof.

  The carrier 531 carries a specific component that produces a component that is dropped through the dropping window 511 and decomposes the biofilm of bacterial cells in response to the dissolved component of the diluent in the sample. For example, if the component that decomposes the biofilm is nitric acid and the diluent is a nitrite solution, the specific component supported on the support 531 is exemplified by an organic acid. In the same case, if the diluting solution is an organic acid solution, the specific component supported on the supporting portion 531 is exemplified by nitrite. In either case, nitric acid is generated when the dissolved component of the diluent in the sample comes into contact with the specific component supported on the support 531, and the bacterial biofilm in the sample is decomposed. The material of the supporting portion 531 is not particularly limited, and specifically, natural fibers such as cotton fiber, hemp fiber, wool fiber, and silk fiber; and artificial fibers such as rayon, Bemberg, acetate, and polyamide-based fiber are preferable. The material of the part 531 is mentioned.

  The preferred concentration range in the solution used for carrying the organic acid or nitrite which is the specific component carried on the carrying part 531 is as described above, but the amount of the solution of the concentration is the same as that of the carrying part 531. It can be appropriately adjusted according to the size. The larger the size of the supporting portion 531, the larger the amount of the solution in which the specific component required for sufficient supporting is dissolved. Considering the size of the detection instrument normally used, about 150 to 200 μL is assumed.

  A neutralizing part 532 provided adjacent to the carrying part 531 carries a component for neutralizing the degradation component of the biofilm produced in the carrying part 531. In the case where the biofilm decomposition component is a strong acid such as nitric acid, a component that neutralizes the biofilm decomposition component near neutrality may be mentioned. Specifically, as described above, boric acid, phosphoric acid, trishydroxymethylaminomethane (THAM: commonly called Tris), and the like can be given. The biofilm degradation component infiltrated from the supporting portion 531 and contacted with the neutralizing component supported by the neutralizing portion 532 is neutralized, and the antibody binds to the specific component of the bacteria to be detected in the subsequent mobile phase. Will be done smoothly. This neutralization part 532 is a selective matter of the present invention, but it is extremely preferable to provide this, particularly when the biofilm decomposition component is a strong acid such as nitric acid.

  The labeled antibody binding part 533 is a means by which a labeled antibody labeled with an antibody (polyclonal antibody or monoclonal antibody) that can bind to a specific component of the target bacteria can move from the binding part 533 in accordance with the infiltration of an aqueous solvent. It is supported. When the mobile phase reaches the labeled antibody binding portion 533, if the target bacteria in which the biofilm is decomposed are present in the sample, an antigen-labeled antibody complex in which the specific antibody and the labeled antibody are bound is formed. Then, it moves toward the detection section 534 together with the unbound labeled antibody. When the detection-target bacteria are not present in the sample, the antigen-labeled antibody complex is not formed, and only the unreacted labeled antibody moves toward the detection unit 534. The label of the labeled antibody is not particularly limited, and examples thereof include gold colloid, platinum colloid, colored or uncolored synthetic latex particles, and colored or uncolored natural rubber latex particles. The labeled antibody binding part 533 is provided between the neutralization part 532 (a support part 531 when no neutralization part is provided) and the following detection part 534. As shown in this example, the neutralization part 532 ( In the case where the neutralizing portion is not provided, it is preferable to provide the neutralizing portion as a substrate surface in contact with the carrying portion 531) or in the vicinity of the substrate surface.

  The detection unit 534 is a site for capturing a conjugate of a specific bacterial cell component and a labeled antibody to reveal a labeled signal. Specifically, a second antibody specific to a specific bacterial cell component is fixedly supported and binds to the bacterial cell component portion in the antigen-labeled antibody complex to capture the complex. Since the complex is collected and captured by the detection unit 534, a label such as a colloidal gold or latex particle becomes apparent there as a signal. By observing this manifested signal through the detection window 512, the presence of the detection target bacteria in the sample can be confirmed. When there is no bacteria to be detected in the sample, it is not an antigen-labeled antibody complex that moves to the detection unit 534 but a free labeled antibody, so that it is not captured by the detection unit 534 and is directly downstream. Move to. Therefore, the label signal does not appear in the detection unit 534.

  The control unit 535 is provided to confirm that the mobile phase has sufficiently reached at least the detection unit 534 regardless of whether or not the detection target bacteria are present in the sample. That is, the control unit 535 has a component for binding them, typically an anti-immunoglobulin antibody, regardless of whether it is an antigen-labeled antibody complex or a free labeled antibody. When the detection target bacteria exist, the antigen-labeled antibody complex leaked without being captured by the detection unit 534 and the free labeled antibody that has moved without binding to the specific bacterial component are captured and labeled signal To reveal. In addition, when there is no detection target bacteria in the sample, the free labeled antibody that has passed through the detection section 534 and moved is captured, and the labeled signal is revealed. When both the detection part 534 and the control part 535 make a signal manifest, it determines with positive. The detection unit 534 determines that the signal is negative when only the control unit 535 reveals the signal without revealing the signal. Then, regardless of whether or not the signal from the detection unit 534 is revealed, if the signal does not appear in the control unit 535, it is determined that the detection form is not normal and is invalid.

  The material constituting the solid-phase substrate surface, which is preferably provided with the detection unit 534 and the control unit 535, is not particularly limited, but is preferably a material suitable for showing antibody loading and signal manifestation. is there. In that sense, a material that can be easily formed into a thin film, for example, a nitrocellulose film, glass fiber, polyethersulfone (PES), cellulose, polyester, polyamide fiber, or the like is preferably used.

  The substrate of the solid phase 53 may be a single layer, but may have a composite configuration in which members having a desired function are combined. For example, the lowermost layer of the substrate surface of the solid phase 53 (the layer including the contact surface with the hook structure 521 ′) can be used as an adhesive tape. This adhesive tape may be a single-sided adhesive tape or a double-sided adhesive tape. When the lowermost layer is a double-sided pressure-sensitive adhesive tape, the solid phase 53 is fitted in the catching structure 521 ′ and bonded and fixed to the structure surface via the lower pressure-sensitive adhesive surface. In the case of a single-sided adhesive tape, the non-adhesive surface is the hook structure 521 'side. In any case, on the pressure-sensitive adhesive surface (upper side) of the lowermost pressure-sensitive adhesive tape, the parts constituting the carrier part 531, the neutralizing part 532, and the labeled antibody binding part 533, the detection part 534, and the control part 535 are provided. The parts provided with are bonded and fixed in an appropriate combination. An example in which the lowermost layer of the substrate of the solid phase 53 is an adhesive tape has been disclosed in Examples described later.

  In this embodiment, the specimen extraction diluent (sample) is directly dropped onto the carrier 531 carrying the specific component. However, the carrier and the dropping part in the solid phase are separately provided. It may be. In such a case, the dropping unit is disposed upstream of the supporting unit 531, and the sample dropped on the dropping unit moves to and contacts the supporting unit as a mobile phase to generate a biofilm decomposition component, which is described above. The target bacteria can be detected by the detection unit 534 according to the background. A drip window 511 is provided at a position corresponding to the drip part separated from the carrier part 531.

  6, FIG. 7, FIG. 8, and FIG. 9 show one mode when the detection method of the present invention is performed using the detection instrument 50 of the present invention. FIG. 6 is also an example 90 of the detection kit of the present invention, and FIGS. 6 to 9 also show an embodiment using the detection kit of the present invention.

  The configuration of the detection kit 90 shown in FIG. 6 is the detection instrument 50, the mixing and dropping instrument 60, and the cotton swab 70 of the present invention described above. As a configuration of the detection kit of the present invention, the detection instrument 50 and the mixing and dropping instrument 60 of the present invention are essential elements, and the swab 70 is an optional element. Further, although not shown in FIG. 6, the specimen dilution liquid is also an essential kit element.

  In FIG. 6, the detection instrument 50 is as described above. The mixing dripping device 60 puts a diluent in the tube, inserts the sample attached to the head 71 of the cotton swab 70 and extracts the sample as a sample extraction diluent (sample). It is an instrument for dropping a sample) from the dropping window 511 of the detection instrument 50 onto the carrier portion 531. The mixing and dropping device 60 includes a tube 61 and a lid 62.

  The tube 61 is preferably composed of a tube main body 611 having flexibility and a connecting member 612 fixed to the opening thereof. In the opening formed in the connection member 612, the lid 62 has a shape that can be fitted to the tube 61. The tube 61 and the lid 62 are fitted in a detachable state. Further, the filter 621 is provided so as to be interposed in the filter inlet 622 provided in the vicinity of the central axis in the fitting direction. The filter 621 is a filter that does not allow the aqueous solvent to pass through in a normal state but allows the aqueous solvent to pass through by applying pressure toward the filter surface. Examples thereof include a nitrocellulose filter and a glass filter having such properties. Is done. The sample dilution liquid may be stored in a vial or the like separate from the mixing and dropping device 60, or the filter inlet 622 of the lid 62 is sealed, and preferably the lid 62 and the tube 61 are opened. As shown in FIG. 7 (1), the joint portion may be stored in advance in the mixing and dropping device 60 in a state where the joint portion is also sealed.

  FIG. 7 is a drawing depicting the state of mixing extraction in the mixing and dropping device that is a component of the detection kit 90 of the present invention. FIG. 7 (1) shows the mixing and dropping device 60 in the form in which the above-described specimen diluent 80 is previously stored in a container. Diluent 80 is defined as “A”. This is due to contact with a specific component “B” (FIG. 8 (1)) carried on the carrying portion of the detection instrument 50 described later, thereby producing one component “B” for generating a degradation component of the biofilm to be detected. A ”indicates that the diluent 80 contains. In one example, if the bacterium to be detected is Streptococcus pyogenes and the degradation component of the biofilm is nitric acid, the component “A and B pair” is “Nitrite and organic acid pair”, or It is one of “a pair of organic acid and nitrite”.

  In FIG. 7 (2), the lid 62 of the mixing and dropping device 60 is removed, and a cotton swab 70 with a specimen attached to the head 71 is inserted from the head through the opening of the tube 61 to dilute the tube 61. A state of contact with the liquid 80 is shown. When the diluting solution 80 is not stored in the mixing and dropping device 60 in advance, the diluting solution 80 is poured into the tube 61 from the outside before the cotton swab 70 is inserted.

  FIG. 7 (3) shows an example of how the specimen is extracted from the diluent 80 in the tube 61. In this example, since the tube 61 is flexible during the extraction operation, for example, by swiping the head 71 of the cotton swab on the wall surface of the tube 61 with the finger b as shown by the arrow c, Extraction can be performed. In this way, the diluent 80 becomes the specimen extraction diluent (sample) 80 '.

  FIG. 8 shows an example of the subsequent detection procedure of the detection target bacteria using the detection kit 90 of the present invention. In FIG. 8 (1), the lid portion 62 is taken as a form in which the lid portion 62 is fitted again into the tube 61 in which the specimen extraction diluent (sample) 80 ′ prepared through FIG. 7 (3) is stored. A state in which the mixing and dropping device 60 is brought close to the dropping window 511 of the detection device 50 and the specimen extraction diluted solution (sample) 80 ′ is dropped toward the dropping window 511 is shown. In this example, the drop is a close drop, but may be a drop in contact with the detection window 511 of the mixing dropping apparatus 60. The specimen extraction diluent (sample) 80 ′ contains the above-described component A, and the support 531 on the solid phase 53 supports the component B. When components A and B come into contact with each other, components that decompose the biofilm of the bacteria to be detected are generated. FIG. 8 (2) shows that the specimen extraction diluent (sample) 80 ′ dropped on the support portion 531 of the solid phase 53 has already described “Generation of biofilm decomposition components and biofilm decomposition in the support portion 531 → Through the process of “neutralization of biofilm degradation component in neutralization section 532 → formation of antigen-labeled antibody complex in labeled antibody binding section 533”, a label signal in detection section 534 that indicates the presence of a detection target bacterium in detection section 534, And the signal which shows the confirmation of the arrival of the mobile phase in the control part 535 has shown a mode that it revealed. The presence or absence of the manifestation of these signals can be visually confirmed through the detection window 512.

  In FIG. 9, the process up to the above-described FIG. 8 (2) is shown by a longitudinal section cut in the direction of the arrow XX described above. The lid 62 of the mixing and dropping device 60 is close to the dropping window 511 of the detection device 50, and the specimen extraction diluent (sample) 80 'is dropped. As described above, the lid 62 may be in contact with the dropping window 511 instead of being close thereto. In any case, when the tube 61 with flexibility is pushed again with fingers or the like, the internal pressure to the filter 621 increases, and the specimen extraction diluted solution (sample) 80 ′ passes through the filter 621 and is injected into the filter. It is dropped into the carrier 531 via the inlet 622. The specimen extraction diluent (sample) 80 ′ containing the dropped component A reacts with the component B carried on the carrying part 531 to generate a biofilm decomposing component and decompose the bacterial biofilm. Then, it moves to the neutralization part 522 as a mobile phase, and the decomposition component is neutralized. The neutralized sample moves as a mobile phase in the direction of the arrow d due to the chromatographic phenomenon, and first contacts the labeled antibody binding portion 533, whereby the labeled antibody supported in a form that can be dissociated from the specific bacterial cell component and The antibody-labeled antibody complex is formed, and when the complex is bound to an antibody specific to a specific bacterial cell component immobilized and supported on the detection unit 534, the label added to the antibody is used as a signal. Realize. Furthermore, the control unit 535 can confirm that the mobile phase has moved normally as a signal by capturing the labeled antibody with the immobilized anti-immunoglobulin antibody. These confirmations can be made through the detection window 512.

  The amount of the specimen extraction diluent (sample) 80 ′ dropped on the carrier 531 is not particularly limited, but is assumed to be about 300 μL to 1 mL based on the size of a general mixing and dropping device 60.

Below, the more concrete Example of this invention is shown.
(1) Preparation of antibody-modified gold colloid coating solution The pH was adjusted by adding 1 mL of 50 mM HEPES buffer (pH 8.0) to 10 mL of a colloidal gold aqueous solution having a diameter of 40 nm, and 1 mL of an anti-streptococcus antibody solution was added and stirred. After standing for 10 minutes, 120 μL of 5% polyethylene glycol 20000 aqueous solution was added and stirred, and then 1.2 mL of 10% sodium caseinate aqueous solution was added and stirred. This solution was immediately centrifuged at 12,000 × g · 4 ° C. for 15 minutes, and then the supernatant was removed. Then, 1 mL of gold colloid-labeled antibody storage solution (containing 10% trehalose and 0.1% sodium caseinate) was added, and an ultrasonic cleaner was added. To redisperse the colloidal gold. Thereafter, the OD at 520 nm was adjusted to 2.0 with a colloidal gold labeled antibody storage solution to obtain a colloidal gold labeled antibody solution.

(2) Preparation of specific component-immobilized pad A 9 mm x 300 mm cotton pad is impregnated with a sodium sulfite aqueous solution or a citric acid aqueous solution having the concentrations shown in Table 1 below, and dried overnight at room temperature to specify the supporting part. A component-immobilized pad was obtained.

(3) Preparation of neutralization component-immobilized pad Neutralization component constituting a neutralization part by impregnating a 9 mm x 300 mm cotton pad with 1M borate buffer (pH 8.3) and drying overnight at room temperature. An immobilization pad was obtained.

(4) Preparation of gold colloid-labeled antibody holding pad A colloidal gold-labeled antibody solution is impregnated into 4 mm x 300 mm flat glass wool and dried overnight at room temperature to obtain a gold colloid-labeled antibody holding pad constituting the labeled antibody binding portion. It was.

(5) Preparation of antibody-immobilized membrane 1 mg / mL anti-streptococcus antibody solution for test line (detection part) and 1 mg / mL control (control part) for nitrocellulose membrane cut to 25 mm x 300 mm The anti-rabbit IgG antibody was applied at 0.75 μL / cm using an inkjet type coating apparatus, and then dried at 50 ° C. for 30 minutes using a dryer to obtain an antibody-immobilized membrane.

(6) Preparation of immunochromatographic slip (solid phase) The above-prepared antibody-immobilized membrane was attached to an 80 mm × 300 mm adhesive sheet. A specific component-immobilized pad was affixed from the end of the adhesive sheet on the test line side, and an antibody-modified gold colloid holding pad was affixed to the antibody-immobilized membrane so as to overlap with the antibody-immobilized membrane. The neutralization component-immobilized pad was brought into contact with the specific component-immobilized pad and further adhered to the gold colloid holding pad in a state of being overlapped by about 1 mm. Further, an absorption pad was laminated and pasted on the control line side of the antibody-immobilized membrane. The laminated members were cut with a guillotine cutter so that the long side of the member was 5.5 mm wide, and an immunochromatographic strip was prepared.

(7) Preparation of immunochromatographic device The immunochromatographic strip prepared as described above was stored in an ABS plastic case to obtain an immunochromatographic device.

(8) Evaluation of immunochromatographic device The streptococcus is obtained by diluting a positive control (1 × 10 ⁸ cells / mL) attached to Elnas Strip A (TFB) with a phosphate buffer to 1 × 10 ⁵ cells. / A was prepared (A). On the other hand, the pharynx was scraped with a cotton swab, and cell debris was extracted from the top of the cotton swab with an extract to prepare a diluted solution (B) for a false positive specimen. The extract (B) is an aqueous solution adjusted so that the citric acid or nitrite concentration (concentration in the whole false positive specimen) becomes the values shown in Table 1 below.
30 μL of the bacterial cell fluid (A) and 270 μL of the extract (B) were collected and mixed, and 300 μL of a pseudopositive specimen having a cell concentration of streptococci containing 1 × 10 ⁴ bacterial cells / mL was obtained. Prepared. This dilution concentration corresponds to the best detection sensitivity of streptococci at present.

  300 μL of this pseudo-positive specimen was sealed in an extraction container, a dropping nozzle was attached to the opening of the extraction container, and about 200 μL was dropped onto the immunochromatography device. About 10 minutes after the dropping, it was judged positive if a red line due to agglutination reaction of colloidal gold that formed an immune complex on the test line could be confirmed, and negative if not confirmed. In addition, if a red line could not be confirmed on the control line, it was judged invalid. In Table 1, “+” is a positive that is apparent to ordinary people, and “±” indicates a light coloration that can be finally recognized by a skilled person as positive. “-” Is clearly negative.

  In Table 1, the horizontal numbers indicate the concentration (M) of the dropped solution (200 μL) marked in the upper frame, and the vertical numbers indicate the specific component fixed in the left frame. The concentration (M) of the activated pad impregnation solution (100 μL) is shown.

  As a result, it was confirmed that the product of the present invention can avoid the complexity of detection of streptococci and that the detection sensitivity is also good, and the very practical utility of the product of the present invention was recognized.

50: Detection instrument 50 'of the present invention: Case 51 of the detection instrument of the present invention: Upper exterior part 511: Dropping window 512: Detection window 52: Lower exterior part 521': Rectangular area (hooking structure)
53: Solid phase 531: Supporting part 532: Neutralizing part 533: Labeled antibody binding part 534: Detection part 535: Control part 54: Concavity and convexity (for gripping)
60: Mixing and dropping device 61: Tube 611: Tube body 612: Connection member 62: Lid 621: Filter 622: Injection port 70: Cotton swab 71: Cotton swab head 80, 80 ': Sample dilution liquid (sample)
90: Detection kit of the present invention

Claims (13)

  A method for detecting a bacterium by detecting a biofilm of a bacterium to be detected on a solid phase on a solid phase and using a signal indicating an antigen-antibody reaction on the solid phase against a bacterium component after the biofilm decomposition, On the solid phase, there is a detection unit for revealing the presence of the bacteria to be detected through the signal of the antigen-antibody reaction, and it reacts with the dissolved component of the diluent in the sample separately from the detection unit. A component that decomposes the biofilm by contacting the sample in which the specific component that produces the component that decomposes the biofilm of the bacterial cell is supported and the specimen is extracted with the diluent. Is produced in the supporting part, the bacterial biofilm in the sample is degraded, and the components of the bacteria in which the biofilm is degraded are subsequently detected by migration in the solid phase. Generated by the contact with the parts, and detecting the detection target bacteria by detection of the signal of antigen-antibody reaction, detection methods of bacteria.   The method for detecting bacteria according to claim 1, wherein the component that degrades the biofilm is nitric acid. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The method for detecting a bacterium according to claim 2.
(1) The diluted solution contains an organic acid, and the specific component is nitrite.
(2) The diluted solution contains nitrite, and the specific component is an organic acid. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The method for detecting a bacterium according to claim 3.
(1) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing is more than 0.04M and less than 0.14M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.06M or less or 0 Or (b) except when the organic acid concentration is 0.12M or more and the nitrite concentration is 0.06M or less).
(2) When the diluent contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluent exceeds 0.60 M but less than 1.4 M, and is supported on the solid phase. The organic acid concentration of the solution used for performing exceeds 0.06M and less than 0.16M (except when the nitrite concentration is 0.80M or less and the organic acid concentration is 0.14M or more) It is. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The method for detecting a bacterium according to claim 3.
(1) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing exceeds 0.06M and less than 0.12M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.10M or more. Is excluded).
(2) When the diluent contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluent exceeds 0.60 M but less than 1.40 M, and is supported on the solid phase. The organic acid concentration of the solution used for performing exceeds 0.06M and less than 0.14M (provided that (a) the nitrite concentration is 0.06M or less and the organic acid concentration is 0.08M or less or 0 .. except for the case of 12M or more).   The method for detecting a bacterium according to any one of claims 1 to 5, wherein the bacterium to be detected is Streptococcus pyogenes.   Detection for performing a method for decomposing a biofilm of a bacterium to be detected on a solid phase, and detecting the bacterium using a signal indicating an antigen-antibody reaction on the solid phase against a component of the bacterium after the biofilm decomposition A detection unit for revealing the presence of bacteria to be detected via an antigen-antibody reaction signal is provided on the solid phase, and a diluent in the sample is upstream of the detection unit. A bacteria detection instrument, comprising a supporting part on which a specific component that produces a component that decomposes a biofilm of bacterial cells in response to the reaction is supported.   The solid phase is disposed and fixed in the case, and a detection window of the case is provided at a position corresponding to the detection unit of the solid phase, and the support unit or a position corresponding to the upstream side of the support unit is diluted with a diluent. The detection instrument according to claim 7, wherein a drip window is provided for dripping the diluted sample into contact with the solid phase.   The detection instrument according to claim 7 or 8, wherein the component that decomposes the biofilm is nitric acid. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The detection instrument according to claim 9.
(1) The diluted solution contains an organic acid, and the specific component is nitrite.
(2) The diluted solution contains nitrite, and the specific component is an organic acid. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The bacteria detection instrument according to claim 10.
(1) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing is more than 0.04M and less than 0.14M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.06M or less or 0 Or (b) except when the organic acid concentration is 0.12M or more and the nitrite concentration is 0.06M or less).
(2) When the diluent contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluent exceeds 0.60 M but less than 1.4 M, and is supported on the solid phase. The organic acid concentration of the solution used for performing exceeds 0.06M and less than 0.16M (except when the nitrite concentration is 0.80M or less and the organic acid concentration is 0.14M or more) It is. The diluent and the specific component are a combination of any of the following (1) and (2), and further, a neutralizing component of nitric acid is supported on the detection unit side of the specific component supporting unit in the solid phase. The bacteria detection instrument according to claim 10.
(1) When the diluting solution contains an organic acid and the specific component is nitrite, the concentration of the organic acid in the diluting solution is more than 0.06M and less than 0.14M, and is supported on the solid phase. The nitrite concentration of the solution used for performing exceeds 0.06M and less than 0.12M (provided that (a) the organic acid concentration is 0.08M or less and the nitrite concentration is 0.10M or more. Is excluded).
(2) When the diluent contains nitrite and the specific component is an organic acid, the nitrite concentration in the diluent exceeds 0.60 M but less than 1.40 M, and is supported on the solid phase. The organic acid concentration of the solution used for performing exceeds 0.06M and less than 0.14M (provided that (a) the nitrite concentration is 0.06M or less and the organic acid concentration is 0.08M or less or 0 .. except for the case of 12M or more). (1) A detection instrument according to claims 7 to 12, (2) a mixing and dropping instrument for mixing a sample and a diluent, and dropping the mixture toward a dropping window of the detection instrument, and (3 ) Bacteria detection kit containing sample dilution.

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