JP5407375B2 - Test tool for oral bacteria and test method for oral bacteria - Google Patents

Description

  The present invention relates to an inspection tool for oral bacteria that can easily determine the number of oral bacteria in a subject, and an inspection method for oral bacteria.

Oral bacteria are considered to cause various diseases such as periodontal disease, tooth decay and bad breath. In recent years, it has become a problem as a cause of pneumonia.
Pathogenic bacteria that cause diseases such as pneumonia are bacteria that are resident in the oral cavity. Therefore, it is desired to keep the oral cavity clean by oral care and reduce the number of oral bacteria sucked into the lungs.

The number of oral bacteria is generally confirmed by a culture method. That is, the inside of the oral cavity is wiped with a sterilized cotton swab, etc., diffused and diluted in the medium, inoculated and cultured, and the number of the grown colonies is counted.
However, such a culture method requires many media and days, is complicated in operation, and further requires a specific facility and technique capable of handling bacteria.

  Therefore, as a method for analyzing bacteria that does not require a specific facility or technology, for example, Patent Document 1 may include a test paper containing a chromogenic enzyme substrate for enzyme activity peculiar to oral microorganisms and oral microorganisms. A method is disclosed in which the number of oral microorganisms is determined by directly contacting a sexual sample and then dropping the color developing solution and observing the color of the test paper with the naked eye.

JP 2005-73650 A

However, since the method described in Patent Document 1 determines the number of oral microorganisms based on the degree of color development, it is easily influenced by the subjectivity of the determiner, and the determination tends to vary.
Further, for example pneumonia, the number of bacteria in the oral cavity has been said that the incidence and at 1 × 10 ⁶ cells / mL or more higher, the number of bacteria in the oral cavity is 1 × 10 ⁶ cells / mL or more instantaneously It was difficult to determine whether or not.
In order to unify the determination or to determine instantaneously, a color sample showing the relationship between the degree of color development of the chromogenic enzyme substrate and the number of bacteria in the oral cavity may be separately prepared and compared. However, for example, when the subject himself / herself makes a determination, the color sample must be at hand at the time of determination, so each person needs to prepare a color sample, which is not practical.

  The present invention has been made in view of the above circumstances, and it is not necessary to compare with a color sample prepared separately, and it is possible to easily determine the number of oral bacteria without being influenced by the subjectivity of the judge. It aims at realization of the inspection tool and the inspection method of bacteria in the mouth.

An inspection tool for oral bacteria according to the present invention is an inspection tool for oral bacteria comprising a test body composed of a plate and a water-absorbing carrier incorporated in the plate. Holding a chromogenic enzyme substrate that develops color by an enzyme activity peculiar to internal bacteria and an enzyme peculiar to oral bacteria, and the plate body has a predetermined color that develops color depending on the number of oral bacteria It is characterized by being colored.
Here, it is preferable that a transparent base material is laminated on one surface of the inspection body.
Moreover, it is preferable that the adhesive layer and the release paper are sequentially laminated on one surface of the inspection body.

Furthermore, the chromogenic enzyme substrate is preferably L-leucine-β-naphthylamide hydrochloride and / or DL-alanine-β-naphthylamide hydrochloride.
In addition, if the plate is colored so that the magenta concentration measured by a Macbeth densitometer is 0.35 to 0.43, the number of bacteria in the oral cavity, which is said to increase the incidence of pneumonia, is determined. Can be judged.
Further, the water-absorbing carrier may contain the chromogenic enzyme substrate or a chromogenic solution that causes the chromogenic enzyme substrate to develop color.

Further, the method for examining oral bacteria according to the present invention comprises dropping a test sample, a chromogenic enzyme substrate, and a chromogenic solution for developing the chromogenic enzyme substrate onto the water-absorbing carrier of the oral bacteria testing tool, And the number of bacteria in the oral cavity is determined by comparing with the color of the plate.
In addition, the method for examining oral bacteria according to the present invention includes a test sample, a chromogenic enzyme substrate, and a chromogenic solution that causes the chromogenic enzyme substrate to develop color on the water-absorbing carrier of the oral bacteria test tool. One that is not contained is dropped, the chromogenic substrate is developed, and the number of oral bacteria is determined by comparison with the color of the plate.

  According to the inspection tool for oral bacteria and the inspection method for oral bacteria of the present invention, it is not necessary to compare with a separately prepared color sample or the like, and the oral bacteria can be easily detected without being influenced by the subjectivity of the judge. The number can be determined.

It is a perspective view which shows an example of the inspection tool of the intraoral bacteria of this invention. It is A-A 'sectional drawing of FIG. It is sectional drawing which shows the other example of the inspection tool of the intraoral bacteria of this invention. It is a perspective view which shows the other example of the inspection tool of the intraoral bacteria of this invention.

Hereinafter, the present invention will be described in detail.
[Inspection tool for oral bacteria]
FIG. 1 and FIG. 2 show an example of an inspection tool for oral bacteria according to the present invention (hereinafter sometimes simply referred to as “inspection tool”). The inspection tool 1 of this example includes an inspection main body 10 including a plate body 11 and a water absorbent carrier 12 incorporated in the plate body 11. 1 is a perspective view of the inspection tool 1, and FIG. 2 is a cross-sectional view taken along line AA 'of FIG. 2-4, the same code | symbol is attached | subjected to the component same as FIG. 1, and the description is abbreviate | omitted.

The water-absorbing carrier 12 is capable of holding a chromogenic enzyme substrate that develops color by enzyme activity specific to oral bacteria and an enzyme specific to oral bacteria.
The water-absorbing carrier 12 is a carrier that has water-absorbing properties and can contain a chromogenic enzyme substrate and a coloring solution described later. Examples of such a carrier include paper, filter paper, water-absorbing polymer, cellulose, non-woven fabric, and cotton.
The shape of the water-absorbing carrier 12 is not limited to FIG. In addition, the thickness of the water-absorbing carrier 12 may be approximately the same as the thickness of the plate 11 described later.

The plate body 11 shown in FIGS. 1 and 2 is provided with a through hole substantially at the center, and is fitted into the through hole so that the water absorbent carrier 12 is fixed. The size and shape of the through hole are not particularly limited, but in the case of a circular shape, the diameter of the circle is preferably about 5 to 20 mm.
Examples of the material of the plate 11 include foamed polystyrene, foamed polyethylene, foamed polypropylene, foamed urethane resin, polystyrene, polyethylene, polypropylene, urethane resin, ABS resin, acrylic resin, polycarbonate and other plastics, paper, wood, metal, and the like. It is done.
The thickness of the plate 11 is not particularly limited, but is preferably 0.5 to 5.0 mm.

The plate 11 is colored in a predetermined color that the chromogenic enzyme substrate develops according to the number of oral bacteria.
The plate 11 only needs to have at least the front surface colored in a predetermined color, but the back surface is preferably colored in the same color. If both surfaces of the plate 11 are colored, the inspection result can be visually observed from both surfaces of the inspection tool 1.
In the present invention, the “predetermined color” refers to, for example, the number of oral bacteria when the incidence of diseases such as pneumonia, periodontal disease, dental caries and bad breath caused by oral bacteria is high. The color (reference color) that the chromogenic enzyme substrate develops in accordance with the reference value.

The degree of color development of the chromogenic enzyme substrate varies depending on the amount (concentration) of the enzyme. Usually, the amount of enzyme increases, that is, the color tends to be strong, that is, darkly colored. In addition, since the number of oral bacteria is proportional to the amount of enzyme, the number of oral bacteria tends to increase as the chromogenic enzyme substrate develops more strongly.
Therefore, when coloring the plate 11 to a predetermined color, the degree of color development (color concentration) and the enzyme concentration (or the number of bacteria in the oral cavity) when the chromogenic enzyme substrate is colored by changing the enzyme concentration. Thus, the color may be colored to match the degree of color development of the chromogenic enzyme substrate.

For example, in the case of pneumonia, it is said that the incidence increases when the number of oral bacteria is 1 × 10 ⁶ / mL or more as described above. Therefore, the color that the chromogenic enzyme substrate develops at the enzyme concentration corresponding to 1 × 10 ⁶ bacteria / mL in the oral cavity is used as a reference color, and the plate 11 is colored in the reference color. Specifically, coloring is performed so that the density of magenta measured by a Macbeth densitometer is 0.35 to 0.43. Thus, as will be described in detail later, when the number of bacteria in the oral cavity is inspected using the inspection tool 1 of the present invention, the oral cavity is colored when the degree of coloration of the chromogenic enzyme substrate is light compared to the color of the plate 11 If the number of internal bacteria is less than 1 × 10 ⁶ / mL and the degree of color development is the same as or darker than the color of the plate 11, the number of oral bacteria may be 1 × 10 ⁶ / mL or more Since it understands, it can be determined instantaneously whether the number of bacteria in the oral cavity is equal to or higher than a reference value for which the incidence of pneumonia increases.

  The plate body 11 may be defined as a reference color as in the case of pneumonia and colored to the reference color, but the present invention is not limited to this. For example, one plate may be colored in a gradation for each color when the chromogenic enzyme substrate develops color according to each enzyme concentration. By coloring in this way, the number of oral bacteria can be determined in more detail.

  The coloring method of the plate body 11 is not particularly limited. For example, after the plate body 11 is molded, the plate body 11 may be colored by applying a paint on the front surface or the back surface of the plate body 11 so as to have a predetermined color. The plate body 11 may be formed by adding a pigment or the like to the raw material of the plate body 11 so as to have a predetermined color.

In the inspection tool 1 shown in FIGS. 1 and 2, the transparent substrate 20 is laminated on one surface of the inspection body 10 directly or via an adhesive layer (not shown). However, when the transparent base material 20 is laminated | stacked on one surface of the test | inspection main body 10, both surfaces of the board 11 shall be colored with the predetermined color.
Although it does not restrict | limit especially as thickness of the transparent base material 20, 50-200 micrometers is preferable.
In the present invention, the term “transparent” refers to a state in which the degree of color development can be visually confirmed when there is no color and the color developing enzyme substrate develops color.

When the transparent base material 20 is directly laminated on the inspection body 10, the material of the transparent base material 20 is, for example, a thermosetting resin such as an acrylic resin, a urethane resin, an epoxy resin, a butyral resin, or a fibrous resin, Photocurable oligomers such as urethane acrylate, epoxy acrylate, polyester acrylate, and polyether acrylate.
On the other hand, when the transparent base material 20 is laminated on the inspection main body 10 via the adhesive layer, examples of the material of the transparent base material 20 include glass, plastic film (for example, cellophane, polyolefin, vinyl chloride resin, etc.). Can be mentioned. In addition, the adhesive constituting the adhesive layer is not particularly limited as long as it is transparent, and examples thereof include epoxy resin-based, acrylic resin-based, and urethane resin-based adhesives. The thickness of the adhesive layer is preferably 50 to 200 μm.

When examining bacteria in the oral cavity, wipe the oral cavity with a sterile cotton swab or the like, and attach the test sample (saliva etc.) to the water-absorbent carrier. An enzyme peculiar to bacteria in a test sample and a chromogenic enzyme substrate are brought into contact with a water-absorbing carrier, and color is developed with a color developing solution, and the degree of color development is compared with the color of the plate 11.
By the way, since the food residue may remain in the oral cavity, the food residue may be collected together with saliva or the like and included in the test sample. When a test sample containing food residue is attached to a water-absorbing carrier and brought into contact with a chromogenic enzyme substrate to cause color development, the portion in contact with the food residue tends to develop a strong color, making it difficult to determine.

However, as shown in FIGS. 1 and 2, if the transparent base material 20 is laminated on one surface of the inspection body 10, the test sample is visually observed from the transparent base material 20 side even if food residue is included. Thus, it is possible to confirm the state of uniform color development and to make determination more easily. This is because the food residue hardly permeates the water-absorbing carrier 12, so that even if the test sample contains food residue, the food residue remains on the surface of the water-absorbent carrier 12, and the components in the test sample other than the food residue are present. It penetrates into the water absorbent carrier 12 and reaches the back surface of the water absorbent carrier 12. Since the water-absorbing carrier 12 contains a chromogenic enzyme substrate and a chromogenic solution, that is, it can penetrate, the chromogenic enzyme substrate and the chromogenic solution also penetrate the water-absorbing carrier 12 and reach the back surface. Therefore, it can be confirmed that the chromogenic enzyme substrate is colored not only on the front surface but also on the back surface of the water-absorbing carrier 12, but since the food residue does not penetrate particularly on the back surface, it can be confirmed that the color is uniformly colored. . Since one surface of the test body 10 corresponds to the back surface of the water-absorbing carrier 12, the state in which the chromogenic enzyme substrate is uniformly colored can be confirmed by viewing the test tool 1 from the transparent substrate 20 side.
In addition, if the transparent base material 20 is laminated | stacked on the one surface of the test | inspection main body 10, the water absorbing carrier 12 will be fixed more firmly to the through-hole of the plate body 11. FIG.

Further, instead of the transparent substrate 20, for example, as shown in FIG. 3, the adhesive layer 30 and the release paper 40 may be sequentially laminated on one surface of the inspection body 10. However, when the adhesive layer 30 and the release paper 40 are sequentially laminated on one surface of the inspection body 10, both surfaces of the plate body 11 are colored in a predetermined color.
Examples of the pressure-sensitive adhesive constituting the pressure-sensitive adhesive layer 30 include acrylic, rubber-based, polyurethane-based, polyester-based, and silicone-based pressure-sensitive adhesives.
Although it does not restrict | limit especially as thickness of the adhesion layer 30, 50-200 micrometers is preferable.

As the release paper 40, various types of paper (Japanese paper, kraft paper, etc.), woven fabric, non-woven fabric, or plastic film (cellophane, polyolefin, vinyl chloride resin, etc.) can be used. Note that the release paper 40 has a smaller peel force with respect to the adhesive layer 30 than the peel force with respect to the adhesive layer 30 of the inspection body 10.
Although it does not restrict | limit especially as thickness of the release paper 40, 50-200 micrometers is preferable.
The adhesive layer 30 and the release paper 40 may be replaced with a commercially available double-sided tape.

If the adhesive layer 30 and the release paper 40 are laminated on one surface of the test body 10, when contacting the enzyme specific to the bacteria in the test sample and the chromogenic enzyme substrate with the water-absorbing carrier at the time of the test. Further, the release paper 40 is peeled to expose the adhesive layer 30, and the inspection tool 1 can be attached to a part of the body such as an arm, hand, chest, armpit, foot, etc., and warmed to a body temperature of around 37 ° C. . As a result, the enzyme reaction can be promoted.
If the adhesive layer 30 is laminated on one surface of the inspection body 10, the water absorbent carrier 12 is more firmly fixed to the through hole of the plate body 11.
Furthermore, if a transparent adhesive or transparent or release paper is used, a state of uniform color can be confirmed by visual observation from the side of the adhesive layer 30 or release paper 40, so even if the test sample contains food residue. Easy to judge.

Here, oral bacteria and a chromogenic enzyme substrate will be specifically described.
Bacteria are roughly classified into Gram-negative bacteria and Gram-positive bacteria by Gram differentiation. Among the gram-positive bacteria, bacteria that are present as many resident bacteria in the oral cavity are lytic bacteria.
Gram-negative bacteria include, for example, Bacteroides sp. Kingella, Moraxella, Brucella, Bordetella, Alcaligenes, Siwanella, Pseudomonas, Agrobacterium, Flavobacterium, Actinobacillus, Pasteurella, Aeromonas, Cardiobacterium, Plesiomonas , Vibrio genus, Hemophilus genus, Gardnerella genus, Butiaxera genus, Sedicia genus, Cytobacter genus, Edward Jera genus, Enterobacter genus, Erwinia genus, Essielihia genus, Funia genus Kurebujera genus Kuruibera genus Moruganera, Proteus, Providencia spp, Salmonella, Serratia, Shigella, include the microorganisms belonging to Tatsumera genus, or Yersinia.
When analyzing these gram-negative bacteria, for example, alanine aminopeptidase can be used as an enzyme unique to gram-negative bacteria in general.

Streptococcus pyogenes, Streptococcus agalactia, Streptococcus equii, Streptococcus disagalacia, Streptococcus zoodemicas, Streptococcus estrostococcus reptosus , Streptococcus pneumoniae, Streptococcus sangis, Streptococcus oralis, Streptococcus morillolam, Streptococcus bovis, Streptococcus equinus, Streptococcus mutans, or Streptococcus salivarius.
When analyzing these septic bacteria, for example, leucine aminopeptidase can be used as an enzyme unique to all streptococci.

  In addition, when analyzing Gram positive bacteria, phosphatase can be utilized as an enzyme peculiar to the whole Gram positive bacteria, for example. Phosphatase is an enzyme that is present in many Gram-positive bacteria and also in some Gram-negative bacteria.

The chromogenic enzyme substrate is a compound that does not develop color before the reaction with the above-described enzyme but develops color for the first time by enzyme activity. In the case of color development, the presence of a color developing solution is necessary. Such a chromogenic enzyme substrate is not particularly limited, and a known chromogenic substance can be used as it is, or a synthetic enzyme substrate obtained by binding a chromogenic substance to an enzyme substrate can be used.
Examples of the coloring substance include p-nitrophenol, o-nitrophenol, p-nitroaniline, β-naphthylamine, and derivatives thereof.

Examples of the substance serving as the enzyme substrate to which the coloring substance is bound include L-leucine and L-alanine.
In order to bind these enzyme substrates to the chromogenic substance, they can be bound by a known means, for example, a covalent bond (for example, a peptide bond, an ester bond, or a glycoside bond). A commercially available synthetic enzyme substrate can also be used.

  Among such chromogenic enzyme substrates, examples of chromogenic enzyme substrates that develop color by enzyme (alanine aminopeptidase) activity specific to Gram-negative bacteria include L-alanine β-naphthylamide, L-alanine p-nitroanilide, L- Alanine-2-amidoacridone, L-alanine 4-trifluoromethyl-7-coumarinamide acetate salt, DL-alanine-β-naphthylamide hydrochloride can be used.

  Examples of chromogenic enzyme substrates that develop color by enzyme (leucine aminopeptidase) activity unique to streptococci include, for example, L-leucine β-naphthylamide, L-leucine p-nitroanilide, L-leucine-2-amidoacridone, and L-leucine. -7-Amido-4-methylcoumarin hydrochloride and L-leucine-β-naphthylamide hydrochloride can be used.

  Examples of chromogenic enzyme substrates that develop color by gram-positive bacteria (enzyme (phosphatase)) activity include, for example, 4-methylumbelliferyl phosphate, 4-trifluoromethylumbelliferyl-phosphate, 7- (3-phenylcoumarin) Nyl) -phosphate, 5-bromo-4-chloro-3-indolyl-phosphate, 4-nitrophenyl-phosphate, 1-naphthyl phosphate, p-nitrophenyl-phosphate, pyridinium-2-methoxy-4- (2-nitrovinyl) ) -Phenyl-phosphate can be used.

  These chromogenic enzyme substrates may be used alone or in combination of two or more. Among these chromogenic enzyme substrates, in particular, L-leucine-β-naphthylamide hydrochloride and / or DL-alanine-β-naphthylamide hydrochloride are difficult to discolor even if left in a cool dark place (about 5 ° C.) for 1 year. Is preferred.

  Examples of the color developing solution for coloring the chromogenic enzyme substrate include p-dimethylaminocinnamaldehyde, 3-methyl-2-benzothiazolinone hydrazone, 1-naphthol-2-sulfonate, and the like.

In the inspection tool of the present invention, the water-absorbing carrier may contain the above-described chromogenic enzyme substrate or coloring solution.
Examples of the method for containing the chromogenic enzyme substrate or the coloring solution in the water-absorbing carrier include a method in which these substances are dissolved in an appropriate solvent and the solution is contained in the water-absorbing carrier. In this case, a solvent that does not affect the performance of the chromogenic enzyme substrate and the chromogenic solution is used. Specifically, N, N-dimethylformamide, dimethyl sulfoxide, ethanol, methanol, acetone, diethyl ether, butanol, phosphate buffer, tris-maleate buffer, purified water, and the like can be used.
If both the chromogenic enzyme substrate and the chromogenic solution are contained in the water-absorbing carrier, the storage stability tends to be lowered, and the chromogenic enzyme substrate may develop color.

  In addition, the inspection tool can be peeled on the inspection main body (however, on the other surface of the inspection main body when a transparent substrate or an adhesive layer is laminated on one surface of the inspection main body). A protective film may be laminated. If the protective film is laminated, it is possible to prevent dirt and the like from adhering to the inspection body. At the time of inspection, the protective film may be peeled off and used. Moreover, if a desorption function is given to the protective film, it can be stored while preventing dirt and the like from adhering to the inspection body, particularly the water-absorbent carrier, during and after the inspection.

  The inspection tool of the present invention is not limited to the one described above. For example, as shown in FIG. 4, the water absorbent carrier 12 may be incorporated so as to be adjacent to the plate body 11.

The inspection tool of the present invention can be manufactured, for example, as follows.
For example, in the case of the inspection tool 1 shown in FIGS. 1 and 2, the plate body 11 is formed so as to have a desired shape, and a paint is applied to both surfaces of the plate body 11 so as to have a predetermined color. Next, a perforation process is performed in the approximate center of the plate 11 to provide a through hole.
Separately, a water-absorbing carrier 12 having a size corresponding to the size of the through-hole provided in the plate 11 is prepared, and fitted into the through-hole of the plate 11 so as to fix the water-absorbing carrier 12, thereby producing the inspection body 10.
Next, the transparent base material 20 is laminated on one surface of the inspection main body 10 via the adhesive layer made of the transparent adhesive described above to obtain the inspection tool 1. Moreover, using the thermosetting resin and photocurable oligomer mentioned above as a material of the transparent base material 20, apply | coating these resin on one side of the test | inspection main body 10, and heat or light-irradiate and harden resin and it is transparent. The substrate 20 may be molded.

In the case of the inspection tool 1 shown in FIG. 3, first, the inspection body 10 is created in the same manner as described above.
Next, an adhesive is applied on one surface of the inspection body 10 to form the adhesive layer 30, and the release paper 40 is laminated on the adhesive layer 30 to obtain the inspection tool 1. Alternatively, an inspection tool 1 may be prepared by separately preparing an adhesive layer formed by applying an adhesive on a release paper, and bonding the inspection body 10 and the inspection body 10 inside. Further, a commercially available double-sided tape may be affixed on one surface of the inspection body 10. However, in this case, it is assumed that release paper is attached to one side of the double-sided tape.

In the case of the inspection tool 1 shown in FIG. 4, the plate body 11 is formed so as to have a desired shape, and paint is applied to both surfaces of the plate body 11 so as to have a predetermined color.
Subsequently, the water absorbing carrier 12 is attached via an adhesive or the like so as to be adjacent to the plate body 11 to create the inspection body 10.
Next, the transparent base material 20 is laminated on one surface of the inspection main body 10 in the same manner as described above to obtain the inspection tool 1.

In addition, when the chromogenic enzyme substrate or the chromogenic solution is included in the water-absorbing carrier, for example, in a solution in which the chromogenic enzyme substrate or the chromogenic solution is dissolved in an appropriate solvent so that the concentration becomes 0.01 to 10% by mass. The water-absorbing carrier is immersed in the water-absorbing carrier so that the water-absorbing carrier contains the solution. Subsequently, it is dried by natural drying, air drying, vacuum drying under reduced pressure, freeze drying or the like.
The amount to be contained in the water-absorbing carrier is not particularly limited. For example, when a solution having a concentration of 0.01 to 10% by mass is used, if 0.5 to 3.0 g of solution permeates 1 g of the water-absorbing carrier. It is enough.

  As described above, the inspection tool of the present invention includes an inspection main body including a water-absorbing carrier and a plate body colored in a color when a chromogenic enzyme substrate that develops color by enzyme activity peculiar to oral bacteria. Therefore, it is possible to easily determine the number of bacteria in the oral cavity without preparing a color sample or the like at the time of inspection and without being compared and without being affected by the subjectivity of the determiner. For example, if a plate colored so that the concentration of magenta is 0.35 to 0.43 is used, it is determined whether or not the number of bacteria in the oral cavity is equal to or higher than a reference value that increases the incidence of pneumonia. Judgment can be made instantly.

[Inspection method for oral bacteria]
Hereinafter, the method for examining bacteria in the oral cavity of the present invention (hereinafter sometimes simply referred to as “test method”) will be described.
The inspection method of the present invention is a method for determining the number of bacteria in the oral cavity using the above-described inspection tool of the present invention. Specifically, a test sample, a chromogenic enzyme substrate, and a chromogenic solution are added dropwise to a water-absorbing carrier of a test tool (provided that a chromogenic enzyme substrate or a chromogenic solution is not included). The color is developed and compared with the color of the plate to determine the number of oral bacteria.

  The test sample is not particularly limited as long as it may contain the bacteria to be examined, but for example, a biological sample derived from the oral cavity (saliva, plaque, etc.) or an oral wipe (pharynx, teeth) , Interdental, gum, lingual, sublingual, or a combination thereof, a wiping solution for the entire oral cavity, and the like.

The test sample can be collected, for example, by strongly wiping the entire oral cavity with a sterile cotton swab or the like for about 4 to 5 turns. The collected test sample may be dropped by applying it directly to the water-absorbing carrier of the test tool, or a sterile cotton swab etc. from which the test sample has been collected can be added in 0.5 mL of sterile phosphate buffer or sterile physiological saline, for example. It is possible to wash out the wipes in the mouth by immersing in 0.02 to 0.2 mL of this liquid with a dropper or the like and drop it on the water-absorbing carrier.
Further, the test sample may be collected by directly licking the water-absorbing carrier. For example, if the inspection tool 1 as shown in FIG. 4 is used, the water-absorbing carrier 12 is not surrounded by the plate body 11 and thus can be easily placed in the oral cavity, and the water-absorbing carrier 12 can be easily licked.

The chromogenic enzyme substrate is usually used after diluting with an appropriate solvent as described above so that the concentration is about 0.01 to 10% by mass. The amount of the diluted solution of the chromogenic enzyme substrate dropped onto the water-absorbing carrier is preferably 0.02 to 0.2 mL.
The timing of dropping the chromogenic enzyme substrate may be before dropping the test sample or after dropping the test sample. However, if the test sample is collected by directly licking the water-absorbing carrier, the chromogenic enzyme substrate is dropped after the collection.

  After dropping the test sample and the chromogenic enzyme substrate on the water-absorbing carrier, it is preferable that the enzyme reaction is allowed to proceed for 5 to 30 minutes. At that time, the enzyme reaction is more likely to proceed if left in a heat insulation chamber or the like maintained at 30 to 40 ° C. Further, for example, using an inspection tool 1 as shown in FIG. 3, the release paper 40 is peeled to expose the adhesive layer 30, and the inspection tool 1 is applied to a part of the body such as an arm, hand, chest, armpit, foot, It may be pasted and warmed at body temperature.

The color developing solution is usually used after diluting with an appropriate solvent as described above so that the concentration is about 0.01 to 10% by mass. The amount of the diluting solution of the color developing solution dropped on the water-absorbing carrier is preferably 20 to 100 μL.
The timing of dropping the color developing solution is not particularly limited, and may be before dropping the test sample and the chromogenic enzyme substrate, or after dropping the test sample and the chromogenic enzyme substrate. Further, it may be dropped between the test sample and the chromogenic enzyme substrate. However, when dropping the color developing solution after dropping the test sample and the chromogenic enzyme substrate, after dropping the test sample and the chromogenic enzyme substrate, the reaction is allowed to proceed for 5 to 30 minutes, and then the color developing solution is dropped. Is preferred.

When the coloring solution is dropped, the chromogenic enzyme substrate develops color due to the enzyme activity specific to the oral bacteria in the test sample, so the number of oral bacteria is determined by comparing the degree of color development with the color of the plate. In addition, since the coloring of the chromogenic enzyme substrate may be weakened when the coloring solution volatilizes, it is desirable to make the determination within 2 to 10 minutes after the coloring.
In particular, if the inspection tool 1 shown in FIGS. 1 and 2 is used, the state of uniform color development can be confirmed by visual observation from the transparent base material 20 side, so that even if the test sample contains food residue, it is easier. Judgment.

In addition, for example, if an inspection tool having a plate body colored so that the concentration of magenta is 0.35 to 0.43 is used, the number of bacteria in the oral cavity increases the incidence of pneumonia. It is possible to instantaneously determine whether or not it is greater than the reference value. That is, when the degree of color development of the chromogenic enzyme substrate is lighter than the color of the plate, the number of oral bacteria in the test sample is less than 1 × 10 ⁶ / mL, and the degree of color development is the color of the plate Can be determined instantaneously that the number of oral bacteria is 1 × 10 ⁶ / mL or more.

The inspection method of the present invention is not limited to the one described above. For example, when using an inspection tool including a water-absorbing carrier containing a chromogenic enzyme substrate or a chromogenic solution, the inspection is performed as follows.
That is, the test sample and the chromogenic enzyme substrate and the liquid that is not contained in the water-absorbing carrier are dropped onto the water-absorbing carrier of the test tool to cause the chromogenic enzyme substrate to develop color and compare with the color of the plate. Determine the number of oral bacteria.

Specifically, when the water-absorbing carrier contains a chromogenic enzyme substrate, first apply the test sample collected with a sterile cotton swab etc. directly to the water-absorbent carrier, or sterilize the sterile cotton swab etc. from which the test sample was collected. Immerse the wipes in the mouth by immersing in phosphate buffer or sterilized physiological saline, drop this solution on a water-absorbent carrier, bring the test sample into contact with the chromogenic enzyme substrate, and leave it for 5 to 30 minutes. Promote enzyme reaction. In that case, it is preferable to leave it in a heat insulation box or the like maintained at 30 to 40 ° C.
Next, a coloring solution diluted so as to have a concentration of about 0.01 to 10% by mass is dropped onto the water-absorbing carrier, and the coloring enzyme substrate is developed to compare the degree of coloring with the color of the plate body. Determine the number of bacteria inside. The coloring solution may be dropped on the water-absorbing carrier before dropping the test sample.

On the other hand, when the coloring liquid is contained in the water-absorbing carrier, first, the test sample is directly applied to the water-absorbing carrier in the same manner as described above, or the liquid from which the wipes in the mouth are washed out is used as the water-absorbing carrier. Or dripping.
Next, the chromogenic enzyme substrate diluted to a concentration of about 0.01 to 10% by mass is dropped onto the water-absorbing carrier, the test sample and the chromogenic enzyme substrate are brought into contact, and allowed to stand for 5 to 30 minutes to promote the enzyme reaction. Let As the enzyme reaction proceeds, the chromogenic enzyme substrate develops color, and when the degree of color development does not change, the number of bacteria in the oral cavity is determined by comparison with the color of the plate. The chromogenic enzyme substrate may be dropped on the water-absorbing carrier before the test sample is dropped.

  As described above, since the inspection method of the present invention uses the above-described inspection tool of the present invention, the number of oral bacteria can be easily determined without comparing with a color sample or the like and without being influenced by the subjectivity of the judge. Can be judged. For example, if an inspection tool provided with a plate colored so that the concentration of magenta is 0.35 to 0.43 is used, the number of bacteria in the oral cavity is equal to or higher than a reference value at which the incidence of pneumonia is increased. Whether or not there is can be determined instantaneously.

  1: Test tool for oral bacteria, 10: test body, 11: plate, 12: water-absorbing carrier, 20: transparent substrate, 30: adhesive layer, 40: release paper.

Claims (8)

An inspection tool for oral bacteria comprising a test body comprising a plate and a water-absorbing carrier incorporated in the plate,
The water-absorbing carrier retains a chromogenic enzyme substrate that develops color by enzyme activity specific to oral bacteria, and an enzyme specific to oral bacteria,
An inspection tool for oral bacteria characterized in that the plate is colored in a predetermined color that the chromogenic enzyme substrate develops according to the number of oral bacteria.   The inspection tool for oral bacteria according to claim 1, wherein a transparent base material is laminated on one surface of the inspection body.   The test tool for oral bacteria according to claim 1, wherein an adhesive layer and release paper are sequentially laminated on one surface of the test body.   The oral bacterium according to any one of claims 1 to 3, wherein the chromogenic enzyme substrate is L-leucine-β-naphthylamide hydrochloride and / or DL-alanine-β-naphthylamide hydrochloride. Inspection tools.   The oral cavity bacterium according to any one of claims 1 to 4, wherein the plate is colored so that a magenta concentration measured by a Macbeth densitometer is 0.35 to 0.43. Inspection tools.   The test device for oral bacteria according to any one of claims 1 to 5, wherein the water-absorbing carrier contains the chromogenic enzyme substrate or a chromogenic solution that causes the chromogenic enzyme substrate to develop color.   A test sample, a chromogenic enzyme substrate, and a chromogenic solution that develops the chromogenic enzyme substrate are dropped onto the water-absorbing carrier of the oral bacteria test tool according to any one of claims 1 to 5 to cause the chromogenic enzyme substrate to develop color. A method for examining oral bacteria, characterized by determining the number of oral bacteria in comparison with the color of the plate.   A test sample and a chromogenic enzyme substrate and a chromogenic solution that develops color of the chromogenic enzyme substrate, which is not contained in the water-absorbing carrier, are dropped onto the water-absorbing carrier of the oral bacteria test tool according to claim 6. And a method for inspecting oral bacteria, characterized in that the chromogenic enzyme substrate is developed and the number of oral bacteria is determined by comparison with the color of the plate.

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