KR20110112384A - Instrument for examining bacteria in oral cavity and method for examining bacteria in oral cavity - Google Patents

Abstract

The present invention includes a test body (10) consisting of a plate body (11) and an absorbent carrier (12) assembled on the plate body (11), and the absorbent carrier (12) is colored by an enzyme activity peculiar to oral bacteria. Oral bacteria and an enzyme specific to the bacteria in the oral cavity, and the plate body 11 is a test tool for the bacteria in the oral cavity which are colored in a predetermined color to which the enzyme is colored according to the number of bacteria in the oral cavity. (One); And a method for testing oral bacteria in the oral cavity for determining the number of bacteria in the oral cavity using the test tool 1 for oral bacteria.

Description

Instrument for Examining Bacteria in Oral Cavity and Method for Examining Bacteria in Oral Cavity

The present invention relates to a test equipment for oral bacteria and a test method for oral bacteria that can easily determine the number of bacteria in the oral cavity of a subject.

This application claims priority based on Japanese Patent Application No. 2009-021902 for which it applied to Japan on February 2, 2009, and uses the content here.

Bacteria in the oral cavity cause various diseases such as gum disease, tooth decay, and bad breath. In recent years, there has also been a problem as a cause of pneumonia.

Pathogenic bacteria that cause diseases such as pneumonia are bacteria that exist in the oral cavity. Therefore, it is desired to keep the oral cavity clean by oral administration and to reduce the number of bacteria in the oral cavity inhaled into the lungs.

It is common to check the number of bacteria in the oral cavity by the culture method. In other words, the oral cavity is wiped with a sterile cotton swab or the like, which is then diffused and diluted in an incubator, inoculated and cultured, and the number of developed bacteria is counted.

However, such a culture method requires many incubators and periods, and requires a specific facility or technique that is complicated to operate and that can handle bacteria.

Thus, as a method for analyzing bacteria which does not require a specific facility or technology, for example, Patent Document 1 includes a test paper containing a colorase substrate for enzyme activity specific to microorganisms in the oral cavity; After direct contact with a sample that may contain microorganisms in the oral cavity, the coloring solution was added dropwise to visually observe the color development of the test paper. A method of determining is disclosed.

Japanese Patent Laid-Open No. 2005-73650

 However, since the method described in Patent Document 1 determines the number of microorganisms in the oral cavity by the strength of the color development degree, it is easy to be subjective to the judge's subjective influence, and deviations are likely to occur in the judgment.

Also, for example, pneumonia was difficult to but say nopahjindago this rate if the number of 1 × 10 ⁶ gae / mL or more of the bacteria the oral cavity, it is determined whether or not instantaneous number of oral bacteria is 1 × 10 ⁶ gae / mL or more .

 In order to unify the judgment or make an instant judgment, a color swatch showing a relationship between the color development of the chromophore substrate and the number of bacteria in the oral cavity may be separately prepared and compared. However, for example, in the case where the examinee himself / herself makes a judgment, each person needs to prepare a color swatch because the color swatch should be around the user at the time of the judgment, which is not practical.

The present invention has been made in view of the above circumstances, and does not need to be compared with a separately prepared color swatch and the like, and is a test tool for oral bacteria and an oral cavity which can easily determine the number of oral bacteria in the oral cavity without being subjective to the judgment of the judge. An object of the present invention is to provide a test method for bacteria.

In order to achieve the above object, the present invention employs the following means.

(1) The present invention is a test instrument for oral bacteria having a test body comprising a plate body and an absorbent carrier assembled to the plate body, wherein the absorbent carrier has an enzyme activity specific to oral bacteria. Color-enzyme substrates that develop by color and enzymes specific to bacteria in the oral cavity; The plate is colored in a predetermined color that the chromophore substrate develops according to the number of bacteria in the oral cavity.

(2) It is preferable that the transparent base material is laminated | stacked on the one side of the said test | inspection body in the test tool as described in said (1).

(3) In the inspection tool described in (1) above, it is preferable that an adhesive layer and a release paper (falling paper) are sequentially stacked on one surface of the inspection body.

(4) The test equipment described in (1) to (3), wherein the chromophore substrate is preferably L-leucine-β-naphthylamide hydrochloride and / or DL-alanine-β-naphthylamide hydrochloride. Do.

(5) It is preferable that the said board body with which the test tool of said (1)-(4) is colored so that the density | concentration of the magenta measured by Macbeth densitometer may be 0.35-0.43. In this case, the number of bacteria in the oral cavity, which is said to increase the incidence of pneumonia, can be determined.

(6) The absorbent carrier provided in the test tool according to (1) to (5) may include a color developing solution for developing the color developing enzyme substrate or the color developing enzyme substrate.

(7) In addition, the present invention is added dropwise to the absorbent carrier of the test tool for bacteria in the oral cavity, the test solution, the color development enzyme substrate and the color development liquid for developing the color development enzyme substrate; By developing chromophore substrates; Determining the number of bacteria in the oral cavity compared to the color of the plate; This is a method for testing bacteria in the oral cavity.

(8) The present invention is also added dropwise to the absorbent carrier of the test tool for bacterial oral cavity, to the test sample, the chromophore substrate and the chromophore which develops the chromophore substrate, which are not included in the absorbent carrier. ; By developing chromophore substrates; Determining the number of bacteria in the oral cavity compared to the color of the plate; It is a test method of oral bacteria.

According to the test tool for oral bacteria and the test method for oral bacteria of the present invention, the number of bacteria in the oral cavity can be easily determined without the need for comparison with a separately prepared color sample and the subjective influence of the judge. .

1 is a perspective view showing an example of a test tool for bacteria in the oral cavity of the present invention.
FIG. 2 is a cross-sectional view taken along line AA ′ of FIG. 1.
3 is a cross-sectional view showing another example of the test equipment for oral bacteria of the present invention.
Figure 4 is a perspective view showing another example of the test equipment for oral bacteria of the present invention.

Hereinafter, the test tool for the intraoral bacteria of the present invention will be described in detail.

1 and 2 show an example of a test tool for the intraoral bacteria of the present invention (hereinafter, simply referred to as "test tool"). The inspection tool 1 of this example is provided with a test body 10 including a plate body 11 and an absorbent carrier 12 assembled to the plate body 11. 1 is a perspective view of the inspection tool 1, and FIG. 2 is a 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 absorbent carrier 12 contains a chromophore substrate that develops color by enzyme activity specific to oral bacteria, and an enzyme specific to oral bacteria.

The absorbent carrier 12 is a carrier having absorbency and capable of absorbing and retaining a colorant substrate and a colorant to be described later. Examples of such a carrier include paper, filter paper, absorbent polymer, cellulose, nonwoven fabric, cotton, and the like.

The shape of the absorbent carrier 12 is not limited to FIG. 1. In addition, the thickness of the absorbent support 12 should just be about the same as the thickness of the board body 11 mentioned later.

In the plate body 11 shown in FIGS. 1 and 2, a through hole is provided almost in the center, and the absorbent carrier 12 is fitted in this through hole. The size or shape of the through hole is not particularly limited, but in the case of a circle, the diameter of the circle is preferably about 5 to 20 mm.

Examples of the material of the plate body 11 include plastics such as expanded polystyrene, expanded polyethylene, expanded polypropylene, expanded urethane resin, polystyrene, polyethylene, polypropylene, urethane resin, ABS resin, acrylic resin, polycarbonate, paper, and wood. And metals.

Although the thickness in particular of the board | plate body 11 is not restrict | limited, 0.5-5.0 mm is preferable.

The plate body 11 is colored with the predetermined color which the said chromophore substrate develops according to the number of bacteria in an oral cavity.

Although at least the surface of the board | substrate 11 should be colored by a predetermined | prescribed color, it is preferable that the back surface is also colored by the same color. If both surfaces of the plate body 11 are colored, the inspection result can be seen visually from both surfaces of the inspection tool 1.

In the present invention, the "predetermined color" refers to the number of bacteria in the oral cavity when the incidence of diseases such as pneumonia, periodontal disease, tooth decay, and bad breath caused by bacteria in the oral cavity becomes high, for example. It is the color (reference color) which a color-enzyme base material develops according to this reference value.

The degree of color development varies depending on the amount (concentration) of the enzyme. Usually, as the amount of enzyme increases, it tends to be stronger, that is, darker in color. In addition, since the number of bacteria in the oral cavity is proportional to the amount of enzyme, the stronger the color of the enzyme, the more the number of bacteria in the oral cavity tends to be.

Therefore, in the case where the plate body 11 is colored in a predetermined color, the relationship between the degree of color development (color concentration) and the enzyme concentration (or the number of bacteria in the oral cavity) when the enzyme concentration is changed to color the enzyme. It is good to obtain and to color in the color tone suitable for the color development of the color development enzyme substrate.

For example, in the case of pneumonia, it is said that the incidence rate becomes high when the number of bacteria in an oral cavity is 1x10 <^6> / mL or more as mentioned above. Therefore, when the number of bacteria in the oral cavity is at an enzyme concentration of 1 × 10 ⁶ cells / mL, the color of the color developing enzyme substrate is used as the reference color, and the plate body 11 is colored using this reference color. Specifically, coloring is carried out so that the concentration of magenta measured by a Macbeth densitometer is 0.35 to 0.43. Accordingly, although the details will be described later, when the number of bacteria in the oral cavity is examined using the test tool 1 of the present invention, when the degree of color development of the chromophore substrate is lighter than the color of the plate 11, When the number of bacteria in the oral cavity is less than 1 × 10 ⁶ cells / mL, and the degree of color development is the same as or darker than the color of the plate body 11, it can be seen that the number of bacteria in the oral cavity is 1 × 10 ⁶ cells / mL or more. Therefore, according to the number of bacteria in the oral cavity, it can be instantly determined whether or not the incidence rate of pneumonia is higher than the reference value.

The plate body 11 may determine the color used as the reference color as in the case of pneumonia, and may be colored in this reference color, but the present invention is not limited thereto. For example, one plate may be colored by gradation in each color when the color-enzyme substrate develops according to each enzyme concentration. In this way, the number of bacteria in the oral cavity can be determined in more detail.

It does not restrict | limit especially as a coloring method of the plate body 11, For example, after shape | molding the plate body 11, you may apply | coat and color a paint on the surface or back surface of the plate body 11 so that it may become a predetermined color tone. Alternatively, the plate body 11 may be molded by adding a pigment or the like so as to have a predetermined color tone in the raw material of the plate body 11.

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 20 is laminated on one surface of the inspection body 10, both surfaces of the plate body 11 are colored in a predetermined color.

Although the thickness of the transparent base material 20 is not specifically limited, 50-200 micrometers is preferable.

In addition, in this invention, "transparence" means the state which can visually confirm the grade of the color development at the time of color development enzyme coloration with no coloration.

When the transparent base material 20 is directly laminated on the inspection body 10, examples of the material of the transparent base material 20 include acrylic resins, urethane resins, epoxy resins, butyral resins, and fibrinous resins. Photocurable oligomers, such as thermosetting resin, urethane acrylate, epoxy acrylate, polyester acrylate, polyether acrylate, etc. are mentioned.

On the other hand, when the transparent base material 20 is laminated | stacked on the test | inspection main body 10 through an adhesive layer, as a material of the transparent base material 20, it is glass, a plastic film (for example, cellophane, polyolefin). And vinyl chloride resins). The adhesive constituting the adhesive layer is not particularly limited as long as it is transparent. Examples of the adhesive include adhesives such as epoxy resins, acrylic resins, and urethane resins. As for the thickness of an adhesive layer, 50-200 micrometers is preferable.

When testing bacteria in the oral cavity, the oral cavity is wiped with a sterile cotton swab or the like, and a test sample (saliva, etc.) is attached to the absorbent carrier. In the absorbent carrier, the bacteria in the sample under test are brought into contact with an enzyme specific to the test sample and a chromophore substrate, followed by color development using a color developing solution, and the degree of color development is compared with the color of the plate body 11.

By the way, since there is a case that the remaining residues (called food residues) of the food remains in the oral cavity, the food residues are included in the test sample collected with saliva and the like. When the test sample including food waste is adhered to the absorbent carrier and developed by contacting with the chromophore substrate, the portion in contact with the food waste tends to be strongly colored. Therefore, color development by the actual number of bacteria in the oral cavity is possible. Determination of the degree may be difficult.

However, as shown in Figs. 1 and 2, when the transparent substrate 20 is laminated on one surface of the test body 10, even if food samples are included in the test sample, the transparent substrate 20 is visible from the side. Since it can confirm the state which developed uniformly, it can determine more easily. Because food residues are less likely to penetrate the absorbent carrier 12, even if the test sample contains food residues, the food residues remain on the surface of the absorbent carrier 12, and the components in the test sample other than the food residues are absorbent. Penetrates into the carrier 12 and reaches the back side of the absorbent carrier 12. Since the absorbent carrier 12 contains a chromophore substrate or a chromophoric solution, that is, it can penetrate therein, the chromophore substrate or the chromic solution also penetrates into the absorbent carrier 12 and reaches the back side. Therefore, it can be seen that the chromophore substrate is colored not only on the surface of the absorbent carrier 12 but also on the back side. In particular, since the food waste does not penetrate from the back side, it can be seen that the color is uniformly formed. Since one side of the test body 10 corresponds to the back side of the absorbent carrier 12, the test tool 1 is visually observed from the transparent substrate 20 side to confirm the state of the color development of the enzyme substrate uniformly. Can be.

In addition, when the transparent base material 20 is laminated on one surface of the inspection body 10, the absorbent carrier 12 is more firmly fixed to the through hole of the plate body 11.

In addition, instead of the transparent base material 20, as shown, for example in FIG. 3, the adhesion layer 30 and the release paper 40 may be laminated | stacked sequentially on one side of the test | inspection main body 10. FIG. However, when the adhesive layer 30 and the release paper 40 are laminated | stacked sequentially on one surface of the test | inspection body 10, both surfaces of the board | substrate 11 are colored by predetermined color.

As an adhesive which comprises the adhesion layer 30, adhesives, such as an acryl type, a rubber type, a polyurethane type, polyester type, a silicone type, are mentioned, for example.

Although the thickness of the adhesion layer 30 is not specifically limited, 50-200 micrometers is preferable.

As the release paper 40, it is possible to use various papers (paper, graft paper, etc.), woven fabrics, nonwoven fabrics or plastic films (cellophane, polyolefin, vinyl chloride resin, etc.). In addition, the peeling force of the release paper 40 on the adhesive layer 30 is smaller than the peeling force on the adhesive layer 30 of the test body 10.

Although the thickness of the release paper 40 is not specifically limited, 50-200 micrometers is preferable.

In addition, the adhesive layer 30 and the release paper 40 may be replaced with a commercial double-sided tape.

When the adhesive layer 30 and the release paper 40 are laminated on one side of the test body 10, the absorbent carrier makes contact with the enzyme and the chromophore substrate specific to the bacteria in the sample to be tested at the time of the test. At this time, the release paper 40 may be peeled off to expose the adhesive layer 30, and the inspection tool 1 may be attached to a part of the body such as an arm, a hand, a chest, an armpit, or a foot, and warmed to a body temperature around 37 ° C. . As a result, the enzyme reaction can be promoted.

In addition, when the adhesive layer 30 is laminated on one surface of the test body 10, the absorbent carrier 12 is more firmly fixed to the through hole of the plate body 11.

In addition, when the transparent adhesive or the transparent release paper is used, the state of uniform color development can be confirmed by visually seeing from the side of the adhesive layer 30 or the release paper 40, so that even if the test sample contains food waste, It can be determined.

Herein, the bacteria in the oral cavity and the chromophore substrate will be described in detail.

Bacteria are roughly classified into gram negative bacteria and gram positive bacteria by gram discrimination. Among the Gram-positive bacteria, bacteria that are abundant in the oral cavity are oral bacteria (stretococcus).

Examples of Gram-negative bacteria include genus Bacterides (except Bacterides burugatas and Bacterides flaziris), genus Preboteta, and genus Beyonera (but Beiro). Genus, genus genus, niceria, genus buranhamera, genus achineta, genus keratera, genus morachisera, genus brella, genus borudetera, alkali genus, Siwanera genus, Pseudomonas genus, Agrobacterium genus, Furabobacterium genus, Actinobacillus genus, Pastyrera genus, Aeromonas genus, Cardiobacterium genus, Presiomonas genus, Vibrio genus, Haemophilus, Gadonerera, Butyaxera, Cedcia, Citrobactera, Edward Gera, Enterobacta, Erwinia, Escherichia, Hajunia, Curebujera, Couruivera Genus, Genus Moruganera, Proteus Genus, Providencia Genus, Salmonel It includes the genus, Serratia genus, Shigeru La, A tajjeu in camera, or El when various microorganisms belonging to the genus California.

When analyzing these gram negative bacteria, an alanine amino peptidase can be used as an enzyme peculiar to all gram negative bacteria, for example.

On the other hand, for example, Streptococcus piogenes, Streptococcus agaractia, Streptococcus equica, Streptococcus disagaractia, Streptococcus zodiemicus, Streptococcus Equis imillis, Streptococcus anginosus, Streptococcus forcinus, Streptococcus uberis, Streptococcus swiss, Streptococcus pneumoniae, Streptococcus caucasus , Streptococcus olaris, Streptococcus molbiram, Streptococcus bovis, Streptococcus equinas, Streptococcus mudans, or Streptococcus sarivarius.

In the case of analyzing these lytic bacteria, for example, leucine amino peptidase can be used as an enzyme specific to all lytic bacteria.

In the case of analyzing Gram-positive bacteria, for example, hostase can be used as an enzyme specific to all Gram-positive bacteria. Hostase is an enzyme that exists in many Gram-positive bacteria and also in some of its Gram-negative bacteria.

A chromophore substrate is a compound which does not develop color before reaction with the enzyme mentioned above, but develops for the first time by enzyme activity. In addition, the presence of a coloring liquid is necessary at the time of color development. Such a chromophore substrate is not particularly limited, and a known chromogenic substance can be used as it is, or a synthetic enzyme substrate in which a chromogenic substance is bound to an enzyme substrate can be used.

As a coloring substance, p-nitro phenol, o-nitro phenol, p-nitro aniline, (beta)-naphthylamine, derivatives thereof, etc. are mentioned, for example.

As a substance used as an enzyme substrate which couples a coloring substance, L- leucine, L-alanine, etc. are mentioned, for example.

In order to bind these enzyme substrates with a coloring substance, it can bind by a well-known means, for example, a covalent bond (for example, a peptide bond, an ester bond, or a glycoside bond etc.). In addition, commercially available synthetic enzyme substrates can also be used.

Among the chromophore substrates, for example, L-alanine β-naphthyl amide and L-alanine p-nitoroanilide as chromophore substrates that are developed by the enzyme (alanine amino peptidase) activity peculiar to Gram-negative bacteria. , L-alanine-2-amide acrylamide, L-alanine 4-trimethyl oomethyl-7-coumarin amide acetate salt, DL-alanine-β-naphthyl amide hydrochloride can be used.

As a chromophore substrate which is developed by the enzyme (leucine amino peptidase) activity peculiar to a lytic bacterium, for example, L-leucine β-naphthyl amide, L-leucine p-nitrolo anirido, L-leucine -2 -Amide amides, L-leucine-7-amide-4methyl coumarin hydrochloride, L-leucine-β-naphthyl amide hydrochloride can be used.

Examples of chromophore substrates developed by Gram-positive bacteria by an enzyme (horatase) activity specific to Gram-positive bacteria include, for example, 4-methyl unberly peryl horsesate, 4-triple-methyl unberry peryl horsesate, 7-(3 -Phenyl coumarinyl)-hoseate, 5-bromo -4-chloro -3-indoryl-hoseate, 4-nitrophenyl-hoseate, 1-naphthyl hoseate, p-nitrophenyl -Hosfate, Pyridinium -2-Methoxy -4-(2-Nitro vinyl)-Phenyl Hosate can be used.

These chromophore substrates may be used individually by 1 type, or may use 2 or more types together. Among these chromophore substrates, they are difficult to discolor even after being left in cold dark place (about 5 ° C) for one year. In particular, L-leucine-β-naphthyl amide hydrochloride and / or DL-alanine-β-naphthyl amide hydrochloride Is preferred.

As a coloring liquid which develops a color-enzyme substrate, p-dimethylamino cinnamic aldehyde, 3-methyl- 2-benzothiazolinone hydrazone, 1-naphthol-2- sulfonate, etc. are mentioned, for example.

In the test tool of the present invention, the above-described color-enzyme substrate or color-coating solution may be included in the absorbent carrier.

As a method of including a coloring enzyme substrate or a coloring solution in an absorbent carrier, for example, a method of dissolving these substances in a suitable solvent and including the solution in the absorbent carrier is mentioned. In this case, a solvent that does not affect the performance of the chromophore substrate and the colorant is used. Specifically, N, N-dimethyl formamide, dimethyl sulfokide, ethanol, methanol, acetone, diethyl ether, butanol, phosphate buffer, tris maleate buffer, purified water and the like can be used.

In addition, when both the chromogenic enzyme substrate and the chromophoric solution are included in the absorbent carrier, the storage stability tends to be inferior, and the chromogenic enzyme substrate may be colored.

In addition, the inspection tool may be laminated with a protective film that can be peeled off on the inspection body (in the case where a transparent base material, an adhesive layer or the like is laminated on one surface of the inspection body) on the other side of the inspection body. If the protective film is laminated, it is possible to prevent dust or the like from adhering to the inspection body. In the case of inspection, you may peel and use a protective film. In addition, if the protective film is provided with a detachable function, the protective film can be stored while preventing dust from adhering to the test body, particularly the absorbent carrier.

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

The inspection tool of the present invention can be produced, 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 molded to have a desired shape, and paint is applied to both surfaces of the plate body 11 so as to have a predetermined color tone. Next, a hole is formed near the center of the plate body 11 to form a through hole.

Separately, the absorbent carrier 12 is prepared in accordance with the size of the through hole provided in the plate body 11, and inserted into the through hole of the plate body 11 to fix the absorbent carrier 12 to make the test body (10).

Next, the transparent base 20 is laminated on one surface of the test body 10 via the adhesive layer composed of the above-mentioned transparent adhesive to obtain the test tool 1. In addition, using the above-mentioned thermosetting resin or photocurable oligomer as a material of the transparent base material 20, these resins are apply | coated on one surface of the test | inspection main body 10, and a resin is hardened | cured by heating or light-irradiating, 20) may be molded.

In the case of the inspection tool 1 shown in FIG. 3, the inspection body 10 is first made in the same manner as the above-described method.

Next, an adhesive is applied on one surface of the test body 10 to form an adhesive layer 30, and the release paper 40 is laminated on the adhesive layer 30 to obtain the test tool 1. In addition, an adhesive coated on a release sheet to form an adhesive layer may be separately prepared, and this and the test body 10 may be attached to each other so that the adhesive layer is inward, so that the test tool 1 may be used. In addition, you may apply a commercial double-sided tape on one surface of the test | inspection body 10. However, in this case, the release paper is stuck 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 shape | molded so that it may become a desired shape, and a coloring material is apply | coated to both surfaces of the plate body 11 so that a predetermined color tone may be colored.

Then, the absorbent carrier 12 is pasted with an adhesive or the like so as to be adjacent to the plate 11 to form the test body 10.

Then, the inspection tool 1 is obtained by laminating the transparent substrate 20 on one surface of the inspection body 10 in the same manner as the above-described method.

In addition, in the case where the absorbent carrier contains a chromophore substrate or a coloring solution, for example, the absorbent carrier is deposited in a solution in which the chromophore substrate or the coloring solution is dissolved in a suitable solvent so as to have a concentration of 0.01-10 mass%. Include the solution in the carrier. Then, drying is performed by natural drying, blowing drying, vacuum drying under reduced pressure, freeze drying, and the like.

The amount to be included in the absorbent carrier is not particularly limited, but for example, when a solution having a concentration of 0.01 to 10% by mass is used, it is sufficient to infiltrate 0.5 to 3.0 g of solution with respect to 1 g of the absorbent carrier.

As described above, the test tool of the present invention includes an absorbent carrier and a test body made of colored plates. The color colored on the plate is the color of the color developing enzyme substrate, which is colored by the enzyme activity peculiar to the oral bacteria. Since the degree of coloration indicates the number of bacteria in the oral cavity, it is not necessary to prepare and compare color samples at the time of examination, and also to easily determine the number of bacteria in the oral cavity without being influenced by the judge's subjectivity. have. For example, by using a plate that has been colored so that the concentration of magenta is 0.35 to 0.43, it is possible to instantly determine whether or not the number of bacteria in the oral cavity is equal to or higher than a standard value of increasing incidence of pneumonia.

Hereinafter, the test method of the oral bacteria (hereinafter, simply referred to as "test method") of the present invention will be described.

The test method of the present invention is a method of determining the number of bacteria in the oral cavity using the test tool of the present invention described above. Specifically, the test sample, the chromophore substrate and the chromophoric solution are added dropwise to the absorbent carrier (including the chromophore substrate or the chromophore) of the test instrument, and the chromophore substrate is developed to develop the color and color of the plate. By comparison, the number of bacteria in the oral cavity is determined.

The sample to be tested is not particularly limited as long as it may contain a bacterium to be examined, but for example, existing biological samples (saliva, plaque, etc.) in the oral cavity, or washing liquids (pharynx, teeth, interdental, Gums, on the tongue, under the tongue, or a combination thereof, washing fluids in the oral cavity, and the like.

The test sample can be collected, for example, by thoroughly wiping the entire oral cavity with a sterile swab for 4 to 5 weeks. The collected test sample may be dropwise applied directly to the absorbent carrier of the test tool. Alternatively, sterile cotton swabs, etc., from which the test sample is taken, are immersed in 0.5 mL of sterile phosphate buffer or sterile saline solution, and washed with the oral wipe, and 0.02 to 0.2 mL of the solution is dropped into an absorbent carrier. Also good.

Alternatively, the sample to be tested may be collected by directly licking the absorbent carrier. For example, when the inspection tool 1 shown in FIG. 4 is used, since the absorbent carrier 12 is not surrounded by the plate body 11, it is easy to put in the oral cavity and the absorbent carrier 12 is easy to lick.

The chromophore substrate is usually used after dilution with a suitable solvent so as to have a concentration of about 0.01 to 10% by mass. The dropping amount (dropping amount) of the dilute solution of the chromophore substrate dropped on the absorbent carrier is preferably 20 to 200 µL.

The dropping timing of the color development enzyme substrate may be either before dropping the test sample or after dropping the test sample. However, if the sample to be tested is collected by directly licking the absorbent carrier, the color development enzyme substrate is dropped after the collection.

After dropping the test sample and the chromophore substrate onto the absorbent carrier, the reaction is preferably left to proceed for 5 to 30 minutes. In that case, if it is left to stand in the warming chamber etc. which were maintained at 30-40 degreeC, enzyme reaction force will advance more easily. For example, using the inspection tool 1 as shown in FIG. 3, the release paper 40 is peeled off to expose the adhesive layer 30, and the inspection tool 1 is placed in the arms, hands, chest, armpits, You can attach it to parts of your body, such as your feet, and warm it by body temperature.

The color developing solution is usually diluted with an appropriate solvent so as to have a concentration of about 0.01 to 10% by mass. The dropwise amount of the dilution liquid of the coloring liquid dropping onto the absorbent carrier is preferably 20 to 100 µL.

The dropping timing of the color developing liquid is not particularly limited, and may be either before dropping the test sample and the color developing enzyme substrate or after dropping the test sample and the color developing enzyme substrate. It may also be added dropwise between the test sample and the drop of the color development enzyme substrate. However, when dropping the colorant solution after dropping the test sample and the colorant enzyme substrate, it is preferable to drop the colorant solution after the test sample and the colorant enzyme substrate are dropped for 5 to 30 minutes to proceed the enzymatic reaction. Do.

When the coloring liquid is added dropwise, the color enzyme substrate is colored by the enzyme activity peculiar to the oral bacteria in the test sample. Therefore, the number of bacteria in the oral cavity is determined by comparing the degree of color development with the color of the plate. In addition, when the coloring liquid is volatilized, there is a possibility that the color development of the color developing enzyme substrate may be weak.

In particular, by using the inspection tool 1 shown in Figs. 1 and 2, it is possible to confirm the state of uniform color development by visually seeing from the transparent substrate 20 side, so that even if food waste is contained in the sample to be tested, It can be determined.

Further, for example, when the test is carried out using a test tool having a colored plate so that the concentration of magenta is 0.35 to 0.43, it can be immediately determined whether or not the number of bacteria in the oral cavity is greater than or equal to the threshold that the incidence of pneumonia increases. Can be. That is, when the degree of color development of the chromophore substrate is light compared to the color of the plate, when the number of oral bacteria in the test sample is less than 1 × 10 ⁶ / mL, and the degree of color development is the same or thicker than the color of the plate, It can be instantly determined that the number of bacteria in the oral cavity is 1 × 10 ⁶ cells / mL or more.

The test method of the present invention is not limited to the above-described one. For example, when a test tool having an absorbent carrier containing a color-enzyme substrate or a color-coating solution is used, the test is carried out as follows.

That is, the absorbent carrier of the test instrument is loaded with the sample to be tested and the one that is not included in the absorbent carrier among the chromogenic enzyme substrates and the chromophoric solution (chromatase substrate or chromophoric solution), and the chromophore substrate is colored to develop the color of the plate. The number of bacteria in the oral cavity is determined by comparison with.

Specifically, in the case where the absorbent carrier contains a chromogenic enzyme substrate, first, a test sample collected with a sterile swab or the like is applied directly to the absorbent carrier, or a sterile swab, etc., from which the test sample is taken, is sterilized in phosphate buffer or sterile physiological. After immersing in saline, rinsing off the oral cavity, the solution is dropped on an absorbent carrier, and the test sample is brought into contact with the chromophore substrate for 5 to 30 minutes to promote the enzymatic reaction. In that case, it is preferable to leave it in the heat storage etc. which were preserve | saved at 30-40 degreeC.

 Subsequently, the coloring solution diluted to a concentration of about 0.01 to 10% by mass is added dropwise to the absorbent carrier, and the coloring enzyme substrate is developed to compare the degree of color development with the color of the plate to determine the number of bacteria in the oral cavity. . In addition, the coloring solution may be added dropwise to the absorbent carrier before dropping the test sample.

On the other hand, when the absorbent carrier contains a color developing solution, the test sample may be applied directly to the absorbent carrier in the same manner as described above, or the liquid washed out of the oral cavity may be dropped on the absorbent carrier.

Then, the dilute chromophore substrate diluted to about 0.01 to 10 mass% is added to the absorbent carrier, and the test sample and the chromophore substrate are left in contact for 5 to 30 minutes to promote the enzymatic reaction. As the enzyme reaction proceeds, the chromophore substrate develops color, and when the degree of color development does not change, the number of bacteria in the oral cavity is determined by comparing with the color of the plate. In addition, the chromophore substrate may be added to the absorbent carrier before dropping the test sample.

As described above, the test method of the present invention uses the test tool of the present invention as described above, and thus does not need to be compared with a color swatch and the like, and furthermore, the number of bacteria in the oral cavity can be easily determined without being influenced by the subject's subjectivity. Can be. For example, by using a test tool provided with a plate that is colored so that the concentration of magenta is 0.35 to 0.43, it is possible to instantaneously determine whether or not the number of bacteria in the oral cavity is higher than or equal to the reference that the incidence of pneumonia increases.

1: Test equipment for oral bacteria 10: Test body
11 plate 12 absorbent carrier
20: transparent substrate 30: adhesive layer
40: release paper

Claims (8)

In the inspection tool for oral bacteria comprising a plate body and a test body consisting of an absorbent carrier sandwiched in the plate body,
The absorbent carrier has a chromogenic enzyme substrate which is developed by the enzyme activity peculiar to the bacteria in the oral cavity, and an enzyme specific to the bacteria in the oral cavity,
The plate is a test tool for oral bacteria in which a predetermined color of the color developing enzyme substrate is colored according to the number of bacteria in the oral cavity. According to claim 1, wherein the test instrument for oral bacteria in which a transparent base material is laminated on one side of the test body. According to claim 1, wherein the test tool for oral bacteria in which the adhesive layer and the release paper is sequentially laminated on one side of the test body. The bacterium test according to any one of claims 1 to 3, wherein the chromatase substrate is L-leucine-β-naphthyl amide hydrochloride and / or DL-alanine-β-naphthyl amide hydrochloride. tool. The test tool according to any one of claims 1 to 4, wherein the plate is colored so that the concentration of magenta measured by a Macbeth densitometer is 0.35 to 0.43. The test kit for oral bacteria according to any one of claims 1 to 5, wherein the absorbent carrier comprises a color developing solution for developing the color developing enzyme substrate or the color developing enzyme substrate. To the absorbent carrier of the test tool for oral bacteria in any one of claims 1 to 5, a test sample, a color enzyme substrate, and a color developing solution for developing the color enzyme enzyme are added dropwise to the color enzyme substrate. Method of testing the bacteria in the oral cavity to determine the number of bacteria in the oral cavity by color development and comparison with the color of the plate body. To the absorbent carrier of the test instrument for oral bacteria of claim 6, a test sample and a colorant which does not contain the absorbent carrier among the colorant for developing the colorant enzyme substrate and the colorant enzyme substrate are added dropwise. An intraoral bacterial test method in which the number of bacteria in the oral cavity is determined by developing a substrate and comparing the color of the plate.

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