Identification method, fluorescence measuring device and test agent

Abstract

The present invention provides a discrimination method, a fluorescence measuring device and a test agent that can easily discriminate the genotype of the pathogen of periodontal disease based on enzyme activity. The discrimination method discriminates the genotype of the pathogen of periodontal disease by irradiating an excitation light onto a liquid sample and discriminating the genotype based on the intensity of the fluorescence emitted from the liquid sample, wherein the liquid sample contains the bacteria or bacterial extract of the pathogen of periodontal disease and a reagent formed by a substrate of an enzyme reaction of fluorescently labeled pathogens, and is adjusted to a pH of 7.0 or above and a pH of 8.5 or below for enzyme reaction. The fluorescence measuring device comprises: an irradiation mechanism that irradiates the liquid sample with excitation light; a detection mechanism that detects the fluorescence emitted from the liquid sample; and a discrimination mechanism that discriminates the genotype based on the intensity of the detected fluorescence. The test agent is used to discriminate the genotype of the pathogen of periodontal disease, and comprises a reagent formed by a substrate of an enzyme reaction of fluorescently labeled pathogens and a pH buffer for dissolving the reagent, and the pH buffer is at a pH of 7.0 or above and a pH of 8.5 or below.

Description

Technical Field

[0001] The present invention relates to a method for distinguishing the genotype of pathogenic bacteria of periodontal disease, a fluorescence measuring device and a testing agent. Background Art

[0002] There are hundreds of types of oral bacteria in the human oral cavity. Among these oral bacteria, three types, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, are classified as the Red Complex, which is highly associated with periodontal disease.

[0003] Periodontal disease is an inflammatory disease caused by oral bacteria, which not only affects periodontal tissues, but is also associated with systemic diseases such as arteriosclerosis in addition to myocardial infarction, diabetes, etc. In the past, periodontal disease was diagnosed by using a probe to check the depth of the periodontal pocket or the presence of bleeding, an X-ray examination to observe the alveolar bone, etc. In addition, as described in Patent Documents 1 and 2, an in vitro diagnostic test agent for checking the presence of pathogens has also been developed.

[0004] In the field of bacteriology and clinical practice, among the three types of bacteria classified as red complex, Porphyromonas gingivalis (Pg bacteria) is considered to have a significant impact on the severity of periodontal disease, that is, the progression of alveolar bone resorption. Pg bacteria have pili on the surface of the bacteria, and it is known that the components of the bacterial surface such as pili are greatly related to the adhesion and colonization of Pg bacteria in the oral cavity.

[0005] As a gene encoding the fimbrial protein of Pg bacteria, there is the fimA gene encoding a subunit of the fimbrial protein. It has been confirmed that the fimA gene shows genetic polymorphism and is classified into five types, I to V (types 1 to 5). In the past, it has been reported that the adhesion ability and colonization ability or pathogenicity of Pg bacteria in the oral cavity may differ depending on the genotype of the fimA gene.

[0006] For example, Non-Patent Document 1 reports that the retention rate of type I fimA gene in adults with healthy periodontal tissue is about 70%, the retention rate of type V is about 30%, and the others are less than about 10%. On the other hand, the retention rate of type II (type 2) in adult periodontitis patients is about less than 60%, the retention rate of type IV (type 4) is about less than 20%, and the others are less than about 10%. It is reported that more than 90% of Pg bacteria detected from patients with advanced periodontitis are type II.

[0007] [Prior art documents]

[0008] [Patent Document]

[0009] Patent Document 1: Japanese Patent Publication No. 2010-130924

[0010] Patent Document 2: Japanese Patent Publication No. 2007-519923 [Non-Patent Document]

[0011] Non-patent document 1: Atsushi Amano, "Relationship between the adhesion ability of Porphyromonas gingivalis pili and the periodontal pathogenicity of gene polymorphisms", Journal of the Japanese Society of Periodontology, 2003, Vol. 45, No. 4, pp. 357-363 Summary of the invention

[0012] [Technical problem to be solved by the invention]

[0013] There is a suggestion that the genotypes of the pathogenic bacteria of periodontal disease may have different effects on the pathology of periodontal disease, just like the fimA gene of Pg bacteria. Based on this, when diagnosing, treating and preventing periodontal disease, if the genotypes of the oral flora of the patient can be determined, it may be possible to appropriately evaluate the pathology of periodontal disease or predict the possibility of aggravation.

[0014] In the past, molecular biological methods such as RT-PCR were used as methods for distinguishing genotypes. However, molecular biological methods are time-consuming and laborious to operate, and difficult to implement simply. In addition, molecular biological methods can indirectly measure the copy number or cell number of genes on the one hand, but on the other hand, they cannot measure the enzyme activity that actually causes periodontal disease to progress.

[0015] Pg bacteria classified as red complex produce gingipain, a protease, etc. These enzymes cause flora disturbances such as dysbiosis or promote inflammatory reactions, which are believed to cause periodontal disease to progress. Therefore, a means of distinguishing genotypes based on direct indicators that can be performed more easily than conventional molecular biological methods and more in line with the pathology of periodontal disease is required.

[0016] Therefore, an object of the present invention is to provide a method for easily distinguishing the genotype of periodontal disease pathogenic bacteria based on enzyme activity, a fluorescence measurement device, and a test agent.

[0017] [Technical solutions for solving technical problems]

[0018] In order to solve the technical problem, the identification method involved in the present invention is a method for identifying the genotype of the pathogen of periodontal disease, which irradiates excitation light onto a liquid sample and identifies the genotype based on the intensity of fluorescence emitted from the liquid sample, wherein the liquid sample contains the pathogen of periodontal disease and a reagent formed by a substrate of an enzyme reaction of fluorescently labeled the pathogen, and is adjusted to a pH of above 7.0 and below 8.5 for an enzyme reaction.

[0019] In addition, the fluorescence measuring device involved in the present invention is a fluorescence measuring device for distinguishing the genotype of the pathogen of periodontal disease, which has an irradiation mechanism, a detection mechanism and a discrimination mechanism, wherein the irradiation mechanism is used to irradiate excitation light to a liquid sample, the liquid sample contains the pathogen of periodontal disease and a reagent formed by a substrate of an enzyme reaction of the pathogen fluorescently labeled, and is adjusted to a pH of above 7.0 and below 8.5 for an enzyme reaction; the detection mechanism is used to detect fluorescence emitted from the liquid sample; and the discrimination mechanism distinguishes the genotype based on the intensity of the detected fluorescence.

[0020] In addition, the test agent involved in the present invention is a test agent for determining the genotype of the pathogen of periodontal disease, which comprises a reagent and a pH buffer, wherein the reagent is a substrate of an enzyme reaction of the pathogen that is fluorescently labeled; the pH buffer dissolves the reagent, and the pH buffer is above pH 7.0 and below pH 8.5.

[0021] [Effects of the invention]

[0022] According to the present invention, a method for easily distinguishing the genotype of periodontal disease pathogenic bacteria based on enzyme activity, a fluorescence measurement device, and a test agent can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flowchart showing the flow of a determination method according to an embodiment of the present invention.

[0024] Figure 2A This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.0) obtained by reacting bacterial enzymes.

[0025] Figure 2B This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.5) obtained by reacting bacterial enzymes.

[0026] Figure 2C This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.0) obtained by reacting bacterial enzymes.

[0027] Figure 2D This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.5) obtained by reacting bacterial enzymes.

[0028] Figure 2E This is a graph showing the results of fluorescence measurement of a liquid sample obtained by causing an enzyme having properties similar to gingipain to react by changing the temperature.

[0029] Figure 3A This is a graph showing the results of fluorescence measurement according to pH value of a liquid sample obtained by causing bacterial enzyme reaction.

[0030] Figure 3B This is a graph showing the results of fluorescence measurement for each strain of a liquid sample obtained by causing bacterial enzyme reaction.

[0031] Figure 4A This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.0) obtained by subjecting a bacterial extract to an enzyme reaction.

[0032] Figure 4B This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.5) obtained by subjecting a bacterial extract to an enzyme reaction.

[0033] Figure 4C This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.0) obtained by subjecting a bacterial extract to an enzyme reaction.

[0034] Figure 4D This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.5) obtained by subjecting a bacterial extract to an enzyme reaction.

[0035] Figure 5 This is a graph showing the results of fluorescence measurement according to pH value of a liquid sample obtained by subjecting a bacterial extract to an enzyme reaction.

[0036] Fig. 6A This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.0) obtained by causing bacterial cells to undergo an enzyme reaction at a changed temperature.

[0037] Figure 6B This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.5) obtained by causing bacterial cells to undergo an enzyme reaction at a changed temperature.

[0038] Figure 6C This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.8) obtained by causing bacterial cells to undergo an enzyme reaction at a changed temperature.

[0039] Fig.6D This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.0) obtained by causing bacterial cells to undergo an enzyme reaction at a changed temperature.

[0040] Fig. 6E This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.5) obtained by causing bacterial cells to undergo an enzyme reaction at a changed temperature.

[0041] Fig. 7A The graph shows the results of fluorescence measurement of a liquid sample (pH 7.0) obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature.

[0042] Figure 7B This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.5) obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature.

[0043] Figure 7C This is a graph showing the results of fluorescence measurement of a liquid sample (pH 7.8) obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature.

[0044] Fig.7D This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.0) obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature.

[0045] Fig. 7E This is a graph showing the results of fluorescence measurement of a liquid sample (pH 8.5) obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature.

[0046] Figure 8 This is a diagram for explaining the principle of using bacterial cells to discriminate the genotype of the pathogenic bacteria of periodontal disease.

[0047] Fig. 9 This is a diagram for explaining the principle of distinguishing the genotype of the pathogenic bacteria of periodontal disease using bacterial cell extracts.

[0048] Fig.10 It is a diagram showing the structure of a fluorescence measuring device according to an embodiment of the present invention.

[0049] Fig.11 This is a diagram showing a schematic configuration of a control device included in the fluorescence measuring device.

[0050] Fig.12 This is a flowchart showing the flow of a discrimination method implemented by the fluorescence measuring device.

[0051] Fig.13 This is a flowchart showing the flow of genotype discrimination processing performed by the fluorescence measurement device. DETAILED DESCRIPTION

[0052] <Identification method and test agent>

[0053] First, refer to the attached Figure 1 A determination method and a test agent according to an embodiment of the present invention will be described.

[0054] The discrimination method involved in this embodiment is a method for discriminating the genotype of the pathogenic bacteria of periodontal disease. In this discrimination method, it is discriminated which genotype the pathogenic bacteria of periodontal disease in the collected sample belongs to among the known gene polymorphisms. The discrimination of the genotype is performed based on the enzyme activity that has been confirmed to be correlated with each genotype. The enzyme activity is evaluated by using a test agent and a fluorescence determination method, wherein the test agent uses a substrate for the enzyme reaction after fluorescence labeling.

[0055] The pathogenic bacteria of periodontal disease as the object of genotype discrimination include Porphyromonas gingivalis (Pg bacteria). As the genotype to be discriminated, type I (type 1), type II (type 2), type III (type 3), type IV (type 4), and type V (type 5) of the polymorphic fimA gene encoding fimbrial protein can be listed. Type Ib, which is a subtype of the fimA gene, belongs to type I.

[0056] In the discrimination method involved in this embodiment, a liquid test agent containing a substrate for an enzyme reaction after fluorescence labeling (fluorescently labeled substrate) is added with a bacterial cell of a periodontal disease pathogen of unknown genotype or a bacterial cell extract thereof (test object) to prepare a liquid sample for fluorescence measurement. The fluorescently labeled substrate is a substrate for a degrading enzyme produced by the pathogen of periodontal disease, and the fluorescent chromophore is dissociated by an enzyme reaction. Since the dissociated fluorescent chromophore emits fluorescence when irradiated with excitation light, the intensity of the fluorescence emitted from the liquid sample becomes an indicator of enzyme activity.

[0057] When there is a correlation between the enzyme activity of the test object and the genotype, the test object can be classified into a certain genotype based on the enzyme activity obtained by fluorescence measurement. If the enzyme is reacted under conditions that make the difference in enzyme activity according to genotype clear, the intensity difference of the fluorescence emitted from the liquid sample will become larger, so the genotype of the test object can be clearly determined.

[0058] In order to distinguish the genotype, the test object used to prepare the liquid sample for fluorescence measurement is the bacteria of the pathogenic bacteria of periodontal disease with unknown genotype or its bacterial extract. As the test object, for example, dental plaque, gingival leaching fluid, etc. collected from the oral cavity of the subject can be used directly, or it can also be used as a suspension or the supernatant of the suspension. Generally speaking, in the oral flora of the human body, etc., a certain genotype of the pathogenic bacteria of periodontal disease exists in a dominant state. Therefore, the bacteria to be tested collected from a certain subject can be classified as a certain genotype in a dominant state.

[0059] The liquid sample for fluorescence measurement can be prepared by adding bacteria contained in dental plaque, gingival leaching fluid, etc. to a liquid test agent containing a fluorescently labeled substrate, or by adding a bacterial extract extracted from the bacteria. In addition, the bacterial extract only needs to contain a degrading enzyme that reacts with the fluorescently labeled substrate, and it can include any of an extract separated from residues by subjecting the bacteria to cell destruction treatment, etc., and a secretion / produced by the bacteria outside the cells.

[0060] Figure 1 This is a flowchart showing the flow of a determination method according to an embodiment of the present invention.

[0061] like Figure 1 As shown, the discrimination method according to the present embodiment includes an enzyme reaction step S10 , a fluorescence measurement step S20 , and a genotype discrimination step S30 .

[0062] In the enzyme reaction step S10, the bacteria of the pathogenic bacteria of periodontal disease with unknown genotype or its bacterial extract (test object) is added to the liquid test agent containing the fluorescently labeled substrate (reagent) and the pH buffer solution to start the enzyme reaction. The fluorescently labeled substrate is a substance formed by fluorescently labeling the substrate of the enzyme reaction of the pathogenic bacteria of periodontal disease with a fluorescent chromophore. The liquid test agent to which the test object is added and the enzyme reaction is started becomes the liquid sample to be measured by the fluorescence measurement method.

[0063] As a fluorescently labeled substrate, it is preferred to use a substance in which the C-terminus of a polypeptide is an L-arginine (Arg) residue and a fluorescent chromophore is bonded to the C-terminus of the Arg residue. According to such a fluorescently labeled substrate, since the C-terminus of the Arg residue can be specifically digested by gingipain, a protease produced by Pg bacteria, the enzyme activity of Pg bacteria can be evaluated.

[0064] The fluorescently labeled substrate can be recognized by the protease produced by the pathogenic bacteria of periodontal disease, and it can have an appropriate amino acid sequence. The polypeptide constituting the fluorescently labeled substrate can be composed of any number of amino acids or any type of amino acids. However, from the perspective of stabilizing the enzyme reaction and fluorescence measurement, the preferred polypeptide length is 3 to 4mer.

[0065] The N-terminus of the polypeptide of the fluorescently labeled substrate may be protected by a protecting group, such as isobutyloxycarbonyl (iBoc), tert-butyloxycarbonyl (Boc), acetyl (Ac), benzoyl (Bz), 9-fluorenylmethoxycarbonyl (Fmoc), and the like.

[0066] Preferably, the fluorescent labeling substrate is bonded with aminomethylcoumarin (AMC) as a fluorescent chromophore. According to AMC, no fluorescence is emitted in the state of amide bonding, and fluorescence is emitted only in the state of dissociation, and high-brightness fluorescence can be obtained. Therefore, enzyme activity can be evaluated by highly sensitive fluorescence measurement.

[0067] It is particularly preferred to use a protective group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (Gly-Gly-Arg-MCA) as a fluorescently labeled substrate. According to such a fluorescently labeled substrate, it is easy to be recognized by gingival gingipain produced by Pg bacteria, and a high brightness suitable for measurement can be obtained by AMC. Therefore, the enzyme activity of Pg bacteria can be evaluated more accurately.

[0068] The liquid sample to be measured for fluorescence is preferably a pH buffer containing any one of trishydroxymethylaminomethane (Tris), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) and 2-[4-(2-hydroxyethyl)piperazinyl]ethanesulfonic acid (HEPES) as a main component. Such a pH buffer can maintain a pH value suitable for gingipain produced by Pg bacteria, so the enzyme activity of Pg bacteria can be accurately evaluated.

[0069] It is particularly preferred that the liquid sample to be measured by fluorescence is a pH buffer solution with trishydroxymethylaminomethane (Tris) as the main component. Specific examples of pH buffer solutions with Tris as the main component include Tris-hydrochloric acid buffer solution, Tris-acetic acid buffer solution, Tris-boric acid buffer solution, Tris-phosphate buffer solution, etc. The enzyme activity of Pg bacteria can be evaluated more accurately using pH buffer solutions with Tris as the main component.

[0070] In the fluorescence measurement step S20, the liquid sample is irradiated with excitation light, and the intensity of the fluorescence emitted from the liquid sample is measured. The liquid sample is a solution of a fluorescently labeled substrate (reagent) formed by a bacterial cell of a periodontal disease pathogen of unknown genotype, or a bacterial cell extract thereof (test object) and a substrate for an enzyme reaction of a fluorescently labeled periodontal disease pathogen. The fluorescently labeled substrate dissociates the fluorescent chromophore under the enzyme reaction of the decomposing enzyme produced by the pathogen of periodontal disease, and therefore, emits fluorescence when irradiated with excitation light of a specified wavelength band. Therefore, when performing fluorescence measurement, the fluorescence intensity corresponding to the enzyme activity and reaction time of the test bacteria can be detected.

[0071] The wavelength of the excitation light irradiated to the liquid sample is preferably 350 nm to 380 nm, more preferably 355 nm to 375 nm, and further preferably 360 nm to 370 nm. With such a wavelength, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, a fluorescence intensity suitable for detection can be obtained.

[0072] The wavelength of the fluorescence for measuring the fluorescence intensity is preferably 410 nm to 475 nm, more preferably 425 nm to 465 nm, further preferably 430 nm to 455 nm, and particularly preferably 435 nm to 450 nm. According to such a wavelength, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, fluorescence can be detected with high sensitivity.

[0073] In addition, it is preferred to detect the fluorescence of the liquid sample after adding the test substance to the liquid test agent containing the fluorescent label substrate and before the fluorescent label substrate is completely decomposed / dissociated by the enzyme reaction. In addition, it is preferred to detect the fluorescence of the liquid sample before the fluorescence decays due to the fluorescence lifetime.

[0074] In the genotype discrimination process S30, the genotype of the pathogenic bacteria of periodontal disease (test bacteria) with unknown genotype is discriminated according to the intensity of the fluorescence emitted from the liquid sample after irradiation with the excitation light. The fluorescence intensity value detected by fluorescence measurement indirectly represents the reaction rate of the enzyme reaction, that is, the ratio of the substrate that has reacted. In addition, the time-dependent change of the fluorescence intensity indirectly represents the reaction speed of the enzyme. Therefore, in the case where the enzyme activity of the pathogenic bacteria of periodontal disease is correlated with the genotype, the genotype can be discriminated according to the index values ​​of these fluorescence intensities.

[0075] Here, refer to the attached Figure 1 The specific principles and methods for identifying the genotypes of pathogenic bacteria of periodontal disease are explained.

[0076] FIG. 2 is a graph showing the results of fluorescence measurement of a liquid sample obtained by reacting a bacterial enzyme. Figure 2A This is the result obtained by allowing the enzyme to react at pH 7.0. Figure 2B This is the result obtained by allowing the enzyme to react at pH 7.5. Figure 2C This is the result obtained by allowing the enzyme to react at pH 8.0. Figure 2D This is the result obtained by allowing the enzyme to react at pH 8.5. Figure 2E This is a graph showing the results of fluorescence measurement of a liquid sample obtained by causing an enzyme having properties similar to gingipain to react by changing the temperature.

[0077] exist Figure 2A to Figure 2D In the figure, the results are shown in which a cell suspension of Pg bacteria was centrifuged to collect a precipitate, a liquid sample containing the precipitate of the bacteria was added, the pH conditions were adjusted, an enzyme reaction was carried out at 37.5°C, and then the fluorescence intensity was measured using a fluorescence measuring device. Figure 2E, the results obtained by measuring the fluorescence intensity when the temperature of a liquid sample to which trypsin having properties similar to gingipain was added was changed within the range of 23 to 45°C are shown.

[0078] Figure 2A to Figure 2D The horizontal axis represents the measurement time [minutes] from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity in a specified unit. The thick line in the figure is the result of Pg bacteria whose genotype of the fimA gene is type I, the thin line is the result of Pg bacteria whose genotype is type II, and the dotted line is the result of Pg bacteria whose genotype is type IV. The auxiliary lines in the figure represent the relay (switching) period on the fluorescence measurement device.

[0079] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples per genotype for the liquid sample was 3.

[0080] like Figure 2A to Figure 2D As shown, it is possible to obtain different results of fluorescence intensity according to the genotype of the fimA gene. The fluorescence intensity values ​​at each measurement time show the following tendency, that is, type IV is higher than type I, and type II is higher than type IV. In particular, when adjusted to pH 8.0 and pH 8.5, the difference in fluorescence intensity values ​​according to genotype is expanded. It can be seen that the enzyme activity is different according to the genotype of the pathogenic bacteria of periodontal disease, and the enzyme activity depends on pH.

[0081] according to Figure 2A to Figure 2D The results shown show that, for the method of adding bacterial cells to a liquid sample containing a fluorescently labeled substrate, when the enzyme is reacted in a liquid sample adjusted to a pH greater than 7.5 and below 8.5, the fluorescence intensity of type II and type IV increases, thereby improving the accuracy of genotype discrimination.

[0082] Figure 2E The horizontal axis represents the temperature [°C] of the liquid sample to which trypsin is added, and the vertical axis represents the time change of the fluorescence intensity per minute. The time change of the fluorescence intensity is the value obtained by measuring the fluorescence at a time point of 10 minutes after the start of the enzyme reaction and converting the result into the change per minute. The plotted points in the figure are the average values ​​of the liquid sample temperature at 23°C, 30°C, 37°C, and 45°C, and the error bars are their maximum and minimum values.

[0083] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples for each temperature of the liquid sample was 3.

[0084] like Figure 2EAs shown, it can be confirmed that trypsin, which has properties similar to gingipain, exhibits temperature dependence. The temporal change in fluorescence intensity increases in the range of 23°C to 37°C, and on the other hand, it exhibits a rapid phenomenon in the range of about 37°C to 45°C. Based on the results using trypsin, it is known that the optimal temperature for the enzyme that decomposes the fluorescent labeled substrate is around 37°C.

[0085] according to Figure 2E The results shown can be said to be based on the properties of trypsin, which has properties similar to gingipain. When the enzyme is allowed to react in a liquid sample with a temperature adjusted to above 25°C and below 40°C, especially to around 37±1°C, the change in fluorescence intensity caused by the enzyme reaction becomes larger, and the accuracy of genotype discrimination is improved.

[0086] Figure 3A This is a graph showing the results of fluorescence measurement according to pH of a liquid sample obtained by causing a bacterial enzyme to react.

[0087] exist Figure 3A In the figure, a cell suspension of Pg bacteria whose fimA gene genotype is type II is centrifuged to collect a precipitate, and a liquid sample containing the precipitate added with the bacteria is adjusted to various pH conditions, and an enzyme reaction is carried out at 37.5°C. Thereafter, the fluorescence intensity is measured using a fluorescence measuring device to obtain the result.

[0088] The horizontal axis in the figure represents the measurement time [minutes] from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity in a predetermined unit. The thick dotted line in the figure represents the results at pH 7.0, the single-dash line represents the results at pH 7.5, the middle dotted line represents the results at pH 7.8, the thin dotted line represents the results at pH 7.9, the thick solid line represents the results at pH 8.0, and the thin solid line represents the results at pH 8.5.

[0089] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples of the liquid sample according to pH was 3.

[0090] like Figure 3A As shown, it is possible to obtain results that the fluorescence intensity varies depending on the pH of the liquid sample. The fluorescence intensity values ​​at each measurement time and the time variation of the initial fluorescence intensity show the following tendency, that is, they are higher at pH 7.8 to 8.5 compared to pH 7.0. It is known that pH 7.9 and pH 8.0 show particularly high fluorescence intensity values, and there is a maximum value near pH 8.0.

[0091] according to Figure 3AThe results shown can be said to indicate that, for the method of adding bacteria to a liquid sample containing a fluorescently labeled substrate, when the enzyme is allowed to react in a liquid sample adjusted to a pH of greater than 7.8 and less than 8.5, especially adjusted to a pH of greater than 7.8 and less than 8.0, the fluorescence intensity becomes further higher and the accuracy of genotype discrimination is further improved.

[0092] Figure 3B This is a graph showing the results of fluorescence measurement for each strain of a liquid sample obtained by causing bacterial enzyme reaction.

[0093] exist Figure 3B In the figure, the cell suspension of each Pg bacteria of different strains is centrifuged to collect the precipitate, and the liquid sample containing the precipitate added with the bacteria is adjusted to a specified pH condition, the enzyme reaction is allowed to proceed at 37.5°C, and then the fluorescence intensity is measured using a fluorescence measuring device. The results are obtained.

[0094] The horizontal axis in the figure represents the measurement time [minutes] from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity of the specified unit. The long dashed line in the figure is the result of 33277 strains whose genotype of the fimA gene is type I, the single-point dashed line is the result of the TDC60 strain whose genotype is type II, the short dashed line is the result of the W83 strain whose genotype is type IV, the thick solid line is the result of 275 strains whose genotype is type II, and the thin solid line is the result of 268 strains whose genotype is type II. The 275 strains and the 268 strains are isolates obtained by separation from dental plaque under the gingival margin (refer to Hiroyuki Asano et al., Journal of Periodontology, 2003, Vol. 74, 9, p. 1355-1360).

[0095] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples per strain of the liquid sample was 1.

[0096] like Figure 3B As shown, the results that the fluorescence intensity differed depending on the genotype of the fimA gene were obtained. However, for the TDC60, 275, and 268 strains whose fimA gene genotype was type II, the fluorescence intensity values ​​at each measurement time and the time variation of the fluorescence intensity tended to be similar despite the difference in strain types.

[0097] according to Figure 3B The results shown show that although the enzymatic activity of the degrading enzyme produced by Pg bacteria varies according to the genotype of the pathogenic bacteria of periodontal disease, it is similar between strains of the same genotype, so the genotype can be distinguished based on the results using the fluorescence intensity of any strain.

[0098] FIG. 4 is a graph showing the results of fluorescence measurement of a liquid sample obtained by subjecting a bacterial extract to an enzyme reaction. Figure 4A This is the result obtained by allowing the enzyme to react at pH 7.0. Figure 4B This is the result obtained by allowing the enzyme to react at pH 7.5. Figure 4C This is the result obtained by allowing the enzyme to react at pH 8.0. Figure 4D This is the result obtained by allowing the enzyme to react at pH 8.5.

[0099] exist Figure 4A to Figure 4D , a cell suspension of Pg bacteria was centrifuged to collect the supernatant, a liquid sample to which the supernatant as the bacterial extract was added was adjusted to various pH conditions, an enzyme reaction was carried out at 37.5°C, and then the fluorescence intensity was measured using a fluorescence measuring device.

[0100] Figure 4A to Figure 4D The horizontal axis represents the measurement time [minutes] from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity in a specified unit. The thick line in the figure is the result of Pg bacteria with type I genotype of the fimA gene, the thin line is the result of Pg bacteria with type II, and the dotted line is the result of Pg bacteria with type IV.

[0101] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples per genotype for the liquid sample was 3.

[0102] like Figure 4A to Figure 4D As shown, it is possible to obtain different results of fluorescence intensity according to the genotype of the fimA gene. The fluorescence intensity values ​​at each measurement time show the following tendency, that is, type IV is higher than type II, and type I is higher than type IV. The time variation of fluorescence intensity also shows the same tendency. In particular, when adjusted to pH 8.0 and pH 8.5, the fluorescence intensity value increases according to the difference in genotype. It can be seen that the enzyme activity is different according to the genotype of the pathogenic bacteria of periodontal disease, and the enzyme activity depends on pH.

[0103] according to Figure 4A to Figure 4D The results shown show that, in the method of adding a bacterial extract to a liquid sample containing a fluorescently labeled substrate, when the enzyme is reacted in a liquid sample adjusted to a pH greater than 7.5 and less than 8.5, the fluorescence intensity of type I becomes high, and the discrimination accuracy of genotypes improves. In addition, it can be said that when the enzyme is reacted in a liquid sample adjusted to a pH of about 8.0, the fluorescence intensity of types I and IV becomes high, and the discrimination accuracy of genotypes improves.

[0104] Figure 5This is a graph showing the results of fluorescence measurement according to pH of a liquid sample obtained by subjecting a bacterial extract to an enzyme reaction.

[0105] exist Figure 5 In the figure, a cell suspension of Pg bacteria whose fimA gene genotype is type I is centrifuged to collect the supernatant, and a liquid sample to which the supernatant is added as a bacterial extract is adjusted to various pH conditions, and the enzyme reaction is carried out at 37.5°C. Thereafter, the fluorescence intensity is measured using a fluorescence measuring device to obtain the results.

[0106] The horizontal axis in the figure represents the measurement time [minutes] from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity in a specified unit. The thick dotted line in the figure represents the result at pH 7.0, the single-point dashed line represents the result at pH 7.5, the dotted line represents the result at pH 7.8, the thick solid line represents the result at pH 8.0, and the thin solid line represents the result at pH 8.5.

[0107] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples of the liquid sample according to pH was 3.

[0108] like Figure 5 As shown, it is possible to obtain results that the fluorescence intensity varies depending on the pH of the liquid sample. The fluorescence intensity value at each measurement time and the time variation of the fluorescence intensity show the following tendency, that is, they are higher at pH 8.0 to 8.5 compared to pH 7.0. It is known that pH 8.0 shows a particularly large time variation, and there is a maximum value near pH 8.0 or above pH 8.0.

[0109] according to Figure 5 The results shown show that, in the method of adding a bacterial extract to a liquid sample containing a fluorescently labeled substrate, when the enzyme reaction is carried out in a liquid sample adjusted to pH 8.0 or higher and pH 8.5 or lower, the fluorescence intensity becomes higher and the genotype discrimination accuracy is further improved.

[0110] FIG. 6 is a graph showing the results of fluorescence measurement of a liquid sample obtained by causing bacterial cells to undergo an enzyme reaction while changing the temperature. Fig. 6A This is the result obtained by allowing the enzyme to react at pH 7.0. Figure 6B This is the result obtained by allowing the enzyme to react at pH 7.5. Figure 6C This is the result obtained by allowing the enzyme to react at pH 7.8. Fig.6D This is the result obtained by allowing the enzyme to react at pH 8.0. Fig. 6E This is the result obtained by allowing the enzyme to react at pH 8.5.

[0111] exist Figure 6A to Figure 6E, the results are shown in which a cell suspension of each genotype of Pg bacteria is centrifuged to collect a precipitate, a liquid sample containing the precipitate added thereto is adjusted to various pH conditions, an enzyme reaction is carried out under various temperature conditions, and then the fluorescence intensity is measured using a fluorescence measuring device.

[0112] Figure 6A to Figure 6E The horizontal axis represents the temperature [°C] of the liquid sample, and the vertical axis represents the time change of the fluorescence intensity per minute. The time change of the fluorescence intensity is a value obtained by measuring the fluorescence after the enzyme reaction starts and converting the result into the change per minute. The plotted points ● in the figure are the results of the 33277 strains with the fimA gene genotype of type I, the plotted points ■ are the results of the TDC60 strain with type II, and the plotted points ▲ are the results of the W83 strain with type IV.

[0113] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples per strain of the liquid sample was 3.

[0114] like Fig. 6A As shown, at pH 7.0, the time variation values ​​of each genotype showed similar values ​​at 4-15°C and 45°C. On the other hand, at 22-37.5°C, the time variation values ​​of type I and type IV showed similar values, but the time variation value of type II was higher than that of type I and type IV.

[0115] like Figure 6B As shown, at pH 7.5, the time change values ​​for each genotype showed values ​​of similar magnitude at 4-15°C and 37.5-45°C. At 22-30°C, the time change values ​​for Type II and Type IV were slightly higher than those for Type I, but the difference in the time change values ​​was not large enough.

[0116] like Figure 6C As shown, at pH 7.8, the time change value of type I shows a tendency to be higher at 22 to 37.5°C than at 4 to 15°C and 45°C. The time change value of type IV shows a tendency to be larger than that of type I at 4 to 45°C, and in particular, shows a significantly higher value than that of type I at 4°C and 30°C. The time change value of type II shows a significantly higher value than that of type I and type IV at 4 to 45°C.

[0117] like Fig.6DAs shown in the figure, at pH 8.0, the time variation value of type I shows a tendency to be lower at 37.5 to 45°C than at 4 to 30°C. The time variation value of type IV shows a tendency to be greater than that of type I at 4 to 45°C, and in particular, shows a value significantly higher than that of type I at 15 to 37.5°C. The time variation value of type II shows a value significantly higher than that of type I and type IV at 4 to 45°C, and shows a significantly high maximum value at 22°C.

[0118] like Fig. 6E As shown, at pH 8.5, the time change value of Type I shows a tendency to be lower at 30 to 45°C than at 4 to 22°C. The time change value of Type IV shows a tendency to be greater than that of Type I at 4 to 37.5°C, and in particular, shows a value significantly higher than that of Type I at 15 to 37.5°C. The time change value of Type II shows a value significantly higher than that of Type I and Type IV at 4 to 45°C, and shows a value significantly higher than that of Type I and Type IV at 15 to 37.5°C.

[0119] according to Figure 6A to Figure 6E The results shown show that, for enzyme-encapsulated bacteria, when the enzyme is reacted in a liquid sample adjusted to a pH greater than 7.5 and below 8.5 and adjusted to 4 to 45°C, the fluorescence intensity of type II is higher than that of type I and type IV, and the discrimination accuracy of type II from type I and type IV is improved. In addition, it can be said that when the enzyme is reacted in a liquid sample adjusted to a pH greater than 7.8 and below 8.5 and adjusted to 15 to 37.5°C, the fluorescence intensity of type IV is higher than that of type I, and the discrimination accuracy of the genotype of type IV from type I and type II is improved. It can be said that at 15 to 37.5°C, mutual discrimination between type I, type II, and type V can be performed with high accuracy.

[0120] FIG. 7 is a graph showing the results of fluorescence measurement of a liquid sample obtained by subjecting a bacterial extract to an enzyme reaction at a changed temperature. Fig. 7A This is the result obtained by allowing the enzyme to react at pH 7.0. Figure 7B This is the result obtained by allowing the enzyme to react at pH 7.5. Figure 7C This is the result obtained by allowing the enzyme to react at pH 7.8. Fig.7D This is the result obtained by allowing the enzyme to react at pH 8.0. Fig. 7E This is the result obtained by allowing the enzyme to react at pH 8.5.

[0121] exist Figure 7A to Figure 7E In the figure, the cell suspension of each genotype of Pg bacteria is centrifuged to collect the supernatant, and the liquid sample to which the supernatant as the bacterial extract is added is adjusted to various pH conditions, the enzyme is reacted under various temperature conditions, and then the fluorescence intensity is measured using a fluorescence measuring device. The results are obtained.

[0122] Figure 7A to Figure 7E The horizontal axis represents the temperature [°C] of the liquid sample, and the vertical axis represents the time change of the fluorescence intensity per minute. The time change of the fluorescence intensity is a value obtained by measuring the fluorescence after the enzyme reaction starts and converting the result into the change per minute. The plotted points ● in the figure are the results of the 33277 strains with the fimA gene genotype of type I, the plotted points ■ are the results of the TDC60 strain with type II, and the plotted points ▲ are the results of the W83 strain with type IV.

[0123] Isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA) was used as a fluorescent labeling substrate. The number of samples per strain of the liquid sample was 3.

[0124] like Fig. 7A As shown, at pH 7.0, the time variation values ​​of each genotype showed similar values ​​at 4-22°C and 37.5°C. On the other hand, at 30°C and 45°C, the time variation values ​​of type IV and type II showed similar values, but the time variation value of type I was higher than that of type IV and type II.

[0125] like Figure 7B As shown, at pH 7.5, the time variation value of type IV shows a tendency to be greater than that of type II at 22-37.5°C, and in particular, shows a significantly higher value than type II at 30°C. The time variation value of type I shows a significantly higher value than that of type IV and type II at 15-45°C.

[0126] like Figure 7C As shown, at pH 7.8, the time variation value of type II shows a tendency to be higher at 15 to 37.5°C compared to 4°C and 45°C. The time variation value of type IV shows a tendency to be greater than that of type II at 22 to 37.5°C, and in particular, shows a value significantly higher than that of type II at 37.5°C. The time variation value of type I shows a significantly higher value than that of type IV and type II at 15 to 45°C, and is significantly higher than that of type IV and type II at 30 to 37.5°C.

[0127] like Fig.7D As shown, at pH 8.0, the time variation value of type II shows a tendency to be lower at 45°C compared to 4-37.5°C. The time variation value of type IV shows a tendency to be greater than that of type II at 22-37.5°C, and in particular, shows a value significantly higher than that of type II at 30-37.5°C. The time variation value of type I shows a significantly higher value than that of type IV and type II at 15-37.5°C, and is significantly higher than that of type IV and type II at 22-37.5°C.

[0128] like Fig. 7E As shown, at pH 8.5, the time variation values ​​of Type II and Type IV show values ​​similar to each other at 4 to 45° C. The time variation value of Type I shows significantly higher values ​​than those of Type IV and Type II at 4 to 45° C., and is particularly higher than Type IV and Type II at 37.5° C.

[0129] according to Figure 7A to Figure 7E The results shown show that, when the enzyme-producing bacterial extract is reacted in a liquid sample adjusted to a pH greater than 7.5 and less than 8.5 and adjusted to 15 to 45° C., the fluorescence intensity of type I is higher than that of type IV and type II, and the discrimination accuracy of type I from type IV and type II is improved. In addition, it can be said that when the enzyme is reacted in a liquid sample adjusted to a pH greater than 7.5 and less than 8.0 and adjusted to 30 to 37.5° C., the fluorescence intensity of type IV is higher than that of type II, and the discrimination accuracy of the genotype of type IV from type I and type II is improved.

[0130] Figure 8 This is a diagram for explaining the principle of using bacterial cells to discriminate the genotype of the pathogenic bacteria of periodontal disease.

[0131] Figure 8 The horizontal axis represents the time from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity. The single-dot chain line in the figure shows a specific example of the fluorescence intensity of a liquid sample containing bacteria (analyte) of periodontal disease pathogens of unknown genotype.

[0132] In addition, the thick solid line in the figure shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of type I, the thin solid line shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of type II, and the dotted line shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of type IV.

[0133] like Figure 8 As shown, when a liquid sample containing a bacterial cell of a periodontal disease pathogen of known genotype is subjected to fluorescence measurement, when the enzyme activity of the pathogen of periodontal disease or the extracellular production ability and secretion ability of the enzyme are different according to the genotype, the fluorescence intensity different according to the genotype is detected. When fluorescence measurement is performed on multiple liquid samples with known genotypes and regression analysis is performed on the results, the representative value of the measurement value group of each genotype can be obtained, as shown in the curves (type I line, type II line, type IV line) in the figure. The measurement value of the fluorescence intensity represents a distribution dispersed within a range of a specified distance from each curve (type I line, type II line, type IV line), forming a measurement value group (cluster) divided by genotype (refer to the shaded area in the figure).

[0134] On the other hand, when fluorescence measurement is performed on a liquid sample containing bacteria of periodontal disease pathogens of unknown genotype, a curve (type I line, type II line, type IV line) close to the representative value of a certain genotype can be obtained as shown by the single-dash line in the figure. Figure 8 In the figure, the measurement result close to the type II line is shown. The test substance collected from the subject is in a state where a certain genotype is dominant, and the degree of enzyme activity or enzyme extracellular production ability and secretion ability is similar to the above-mentioned ability of a certain genotype, so such a measurement result can be obtained.

[0135] Therefore, when a predetermined reaction time has passed after the enzyme reaction has started (for example, time T 1 ), the fluorescence intensity of the liquid sample is measured, and the fluorescence intensity values ​​of the liquid sample containing bacteria of periodontal disease pathogens of unknown genotype are compared at the same reaction time (for example, the fluorescence intensity I 0 ) and the fluorescence intensity value of a liquid sample containing bacteria of periodontal disease pathogens of known genotype (e.g., fluorescence intensity I 2 ,I 4 or the boundary line of the shaded area in the figure), the genotype of the object to be tested can be determined.

[0136] In the case of such a discrimination method, when comparing a test area containing a test object with an unknown genotype with a control area containing bacteria of periodontal disease pathogens with a known genotype, although the reaction conditions of the enzyme reaction and the measurement system of the fluorescence measurement need to be consistent, the genotype can be discriminated by simply comparing the fluorescence intensity values.

[0137] Alternatively, if after the enzyme reaction starts, the enzyme reaction is carried out at a predetermined time interval as time passes (e.g., time T 1 The fluorescence intensity of the liquid sample is measured in a small interval near T, and the time-dependent change (slope) of the fluorescence intensity is obtained as the time differential of the fluorescence intensity. The time-dependent change of the fluorescence intensity of the liquid sample containing bacteria of periodontal disease pathogens of unknown genotype (for example, at T 1 -I 0 and the time-dependent change in fluorescence intensity of a liquid sample containing bacteria of periodontal disease pathogens of known genotype (for example, at T 1 -I 2 The slope of the tangent line at the intersection point is 1 -I 4 The genotype of the object to be tested can be determined by the slope of the tangent line at the intersection of the graph or the slope of the tangent line of the boundary line of the shaded area in the figure).

[0138] In the case of such a discrimination method, although it is necessary to calculate the change over time (slope), it is not necessarily necessary to make the reaction time of the enzyme reaction consistent, and the result of the fluorescence measurement is not prone to measurement system errors, so accurate comparison can be made. In addition, the change over time (slope) of the fluorescence intensity can be compared at the same reaction time, and the maximum values ​​in the enzyme reaction can also be compared with each other.

[0139] Fig. 9 This is a diagram for explaining the principle of distinguishing the genotype of the pathogenic bacteria of periodontal disease using bacterial cell extracts.

[0140] Fig. 9 The horizontal axis represents the time from the start of the enzyme reaction, and the vertical axis represents the fluorescence intensity. The single-dot chain line in the figure shows a specific example of the fluorescence intensity of a liquid sample containing a bacterial extract (analyte) extracted from periodontal disease pathogens of unknown genotype.

[0141] In addition, the thick solid line in the figure shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of II, the thin solid line shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of I, and the dotted line shows a specific example of the representative value of the fluorescence intensity obtained from Pg bacteria with a genotype of IV.

[0142] like Fig. 9 As shown, when a liquid sample containing a bacterial extract extracted from a periodontal disease pathogen of known genotype is subjected to fluorescence measurement, when the enzyme activity of the periodontal disease pathogen differs according to the genotype, the fluorescence is not significantly different from that when the bacterial extract is used (refer to Figure 8 ) are the same, and different fluorescence intensities are detected according to genotypes. However, when using bacterial extracts, the influence of the extracellular production ability and secretion ability of the enzyme is reduced, so the measured value of the fluorescence intensity of each genotype shows a tendency different from that when using bacterial cells.

[0143] On the other hand, when the liquid sample containing the bacterial extract extracted from the pathogenic bacteria of periodontal disease of unknown genotype is subjected to fluorescence measurement, as shown by the single-dot chain line in the figure, the measurement result of the curve (type I line, type II line, type IV line) close to the representative value of a certain genotype can be obtained. In the case of using the bacterial extract, the fluorescence intensity of type I and type II becomes the opposite relationship with respect to the case of using the bacterial body, so the smaller the measured value of the fluorescence intensity, the closer it is to the curve of type II, and the larger the measured value of the fluorescence intensity, the closer it is to the curve of type I.

[0144] Therefore, compared with the case of using bacterial cells (refer to Figure 8 ), when using a bacterial extract, after a predetermined reaction time has elapsed after the start of the enzyme reaction (for example, time T1 ), the fluorescence intensity of the liquid sample is measured, and the fluorescence intensity values ​​of the liquid sample containing the bacterial extract extracted from the pathogenic bacteria of periodontal disease of unknown genotype are compared at the same reaction time (for example, the fluorescence intensity I 0 ) and the fluorescence intensity value of a liquid sample containing a bacterial extract extracted from periodontal disease pathogens with known genotypes (e.g., fluorescence intensity I 2 ,I 4 or the boundary line of the shaded area in the figure), the genotype of the object to be tested can be determined.

[0145] In addition, if the enzyme reaction is started at a predetermined time interval and then the enzyme reaction is started, the enzyme reaction is started at a predetermined time interval and then the enzyme reaction is started. 1 The fluorescence intensity of the liquid sample is measured in a small interval near T, and the time-dependent change (slope) of the fluorescence intensity as the time differential is obtained. The time-dependent change of the fluorescence intensity of the liquid sample containing the bacterial extract extracted from the pathogenic bacteria of periodontal disease of unknown genotype is compared (for example, at T 1 -I 0 and the time-dependent change in fluorescence intensity of a liquid sample containing a bacterial extract extracted from periodontal disease pathogens of known genotypes (for example, at T 1 -I 2 The slope of the tangent line at the intersection point is 1 -I 4 The genotype of the object to be tested can be determined by the slope of the tangent line at the intersection of the graph or the slope of the tangent line of the boundary line of the shaded area in the figure).

[0146] For example, the genotype discrimination in the genotype discrimination step S30 can be performed by the following method: preparing a liquid sample in a test area and a liquid sample in a control area, and comparing the fluorescence measurement result of the liquid sample in the test area with the fluorescence measurement result of the liquid sample in the control area, wherein the liquid sample in the test area is composed of a liquid sample containing a pathogenic bacterium of periodontal disease or its bacterial extract (test object) of unknown genotype, and the liquid sample in the control area is composed of a liquid sample containing a pathogenic bacterium of periodontal disease or its bacterial extract of known genotype. In addition, the pH value, temperature, the test object of the pathogenic bacterium or bacterial extract of periodontal disease, the concentration of the fluorescent labeled substrate, etc. of the liquid sample in the test area and the liquid sample in the control area are adjusted to substantially the same conditions and the enzyme reaction is carried out, and then the fluorescence measurement result is obtained for discrimination.

[0147] The control area may be composed of any number of liquid samples, but preferably, it is composed of a plurality of liquid samples. The control area may include the following liquid samples: a liquid sample containing a pathogenic bacterium of periodontal disease with a known genotype or a bacterial extract thereof, and a liquid sample containing a pathogenic bacterium of periodontal disease with an unknown genotype or a bacterial extract thereof. However, from the perspective of reliably distinguishing the genotype, it is preferred that the control area is composed only of a liquid sample containing a pathogenic bacterium of periodontal disease with a known genotype or a bacterial extract thereof.

[0148] The control area may contain a single genotype of bacteria or its extract as the pathogenic bacteria of periodontal disease with a known genotype or its extract, or may contain multiple genotypes of bacteria or its extract as the pathogenic bacteria of periodontal disease with a known genotype or its extract. For example, when the control area contains only type I, it can be determined whether the bacteria to be tested are type I. When the control area contains types I to V, it can be determined which genotype the test object is.

[0149] A control area can be prepared using bacteria or bacterial extracts of strains whose genotypes have been previously identified by fluorescence measurement, or bacteria or bacterial extracts of preserved strains / isolated strains that are generally available through distribution, as periodontal disease pathogens or bacterial extracts whose genotypes are known.

[0150] Specific examples of preserved strains / isolated strains include ATCC_33277 strain with type I fimA gene, ATCC_BAA-1703 (FDC381) strain, JCM_19600 (TDC60) strain, 275 strain (HG184 strain), 268 strain, ATCC_49417 (RB22D-1) strain with type III, ATCC_BAA-308 (W83) strain with type IV, ATCC_53978 (W50) strain, and HNA99 strain with type V.

[0151] Preferably, before the fluorescence measurement, the liquid sample of the fluorescence measurement object is adjusted to a pH greater than 7.5 and below pH 8.5 to allow the enzyme reaction to proceed. The pH value of the liquid sample is preferably above pH 7.8. In addition, the pH value of the liquid sample is preferably below pH 8.4, more preferably below pH 8.3, further preferably below pH 8.2, and further preferably below pH 8.1. If adjusted to such a pH value, the genotype of the fimA gene of the Pg bacteria can be more accurately determined.

[0152] It is preferred that the liquid sample to be measured for fluorescence is subjected to constant temperature control adjusted to a specified temperature. The temperature of the liquid sample is preferably above 4°C and below 45°C. When a bacterial extract is used, the temperature of the liquid sample is preferably above 25°C, more preferably above 30°C, further preferably above 34°C, and further preferably above 36°C. In addition, it is preferably below 40°C, more preferably below 39°C, and further preferably below 38°C. On the other hand, when bacterial cells are used, the temperature of the liquid sample is preferably above 4°C, more preferably above 10°C, further preferably above 15°C, further preferably above 18°C, and further preferably above 21°C. In addition, it is preferably below 37°C, more preferably below 30°C, further preferably below 26°C, and further preferably below 23°C. If controlled at such a temperature, the genotype of the fimA gene of the Pg bacteria can be accurately determined.

[0153] Specifically, the comparison of the fluorescence measurement results of the test area and the fluorescence measurement results of the control area can be performed by comparing the fluorescence intensity value or the time-varying amount of the fluorescence intensity of the liquid sample containing the pathogenic bacteria of periodontal disease or the bacterial extract (test object) of unknown genotype obtained by measuring the test area with the fluorescence intensity value or the time-varying amount of the fluorescence intensity of the liquid sample containing the pathogenic bacteria of periodontal disease or the bacterial extract of known genotype obtained by measuring the control area. When making the comparison, the fluorescence intensity values ​​or the time-varying amounts of the fluorescence intensity are compared with each other.

[0154] The result of comparing the fluorescence measurement results of the test area with those of the control area, when the fluorescence intensity value or the time-dependent change in fluorescence intensity of the liquid sample containing the periodontal disease pathogen or bacterial extract (test object) of unknown genotype obtained by measuring the test area is the same as or similar to the fluorescence intensity value or the time-dependent change in fluorescence intensity of the liquid sample containing the periodontal disease pathogen or bacterial extract of known genotype obtained by measuring the control area, it can be determined that the fimA gene of the periodontal disease pathogen in the liquid sample in the test area and the fimA gene of the periodontal disease pathogen of known genotype in the liquid sample in the control area are of the same genotype.

[0155] On the other hand, when the fluorescence measurement results of the test area are compared with the fluorescence measurement results of the control area, if the fluorescence intensity value or the time-dependent change in fluorescence intensity of the liquid sample containing the periodontal disease pathogen or bacterial extract (test object) of unknown genotype obtained by measuring the test area is different from and not similar to the fluorescence intensity value or the time-dependent change in fluorescence intensity of the liquid sample containing the periodontal disease pathogen or bacterial extract of known genotype obtained by measuring the control area, it can be determined that the fimA gene of the periodontal disease pathogen in the liquid sample in the test area and the fimA gene of the periodontal disease pathogen of known genotype in the liquid sample in the control area are of different genotypes.

[0156] The comparison of the fluorescence measurement results of the test area and the fluorescence measurement results of the control area can be performed, for example, by comparing the measurement values ​​themselves or by comparing representative values ​​such as average values. For example, the measurement values ​​obtained by measuring the test area and the representative values ​​such as average values ​​obtained by measuring the control area are compared. When the difference between the measurement value of the test area and the representative value of the control area is within the range of ±30% relative to the representative value of the control area, the fluorescence measurement results can be judged to be similar to each other.

[0157] Alternatively, the genotype discrimination in the genotype discrimination step S30 can also be performed by the following method: preparing a sample group consisting of a plurality of liquid samples including a liquid sample to be discriminated, and comparing the fluorescence measurement results of the liquid samples in the sample group, wherein the liquid sample to be discriminated includes a pathogenic bacterium or bacterial extract of periodontal disease of unknown genotype (test object). In addition, the pH value, temperature, the amount of the pathogenic bacterium or bacterial extract of periodontal disease to be measured, the concentration of the fluorescent labeled substrate, etc. of the liquid sample to be discriminated and other liquid samples are adjusted to substantially the same conditions, and the enzyme reaction is carried out, and then the fluorescence measurement result is obtained and used for discrimination.

[0158] The sample group may be composed of any number of liquid samples of more than two, but preferably consists of a plurality of liquid samples. The sample group only needs to include the following liquid samples of the discrimination object, wherein the liquid samples of the discrimination object include the pathogenic bacteria or bacterial extract of periodontal disease with unknown genotype (test object), and the sample group may only consist of the liquid sample containing the pathogenic bacteria or bacterial extract of periodontal disease with unknown genotype, or may be composed of the combination of the liquid sample containing the pathogenic bacteria or bacterial extract of periodontal disease with unknown genotype and the liquid sample containing the pathogenic bacteria or bacterial extract of periodontal disease with known genotype.

[0159] It is preferred that the sample group contains strains or bacterial extracts of multiple genotypes as the pathogenic bacteria or bacterial extracts of periodontal disease whose genotypes are known. For example, when the control area contains types I to V, the fluorescence measurement results produce five measurement value groups (clusters) whose tendency of temporal changes in fluorescence intensity differs by genotype. Therefore, by determining the similarity of the fluorescence measurement results of the object to be tested with these measurement value groups, it is possible to determine which genotype the object to be tested is.

[0160] Preferably, before the fluorescence measurement, the liquid sample of the fluorescence measurement object is adjusted to a pH greater than 7.5 and below pH 8.5 to allow the enzyme reaction to proceed. The pH value of the liquid sample is preferably above pH 7.8. In addition, the pH value of the liquid sample is preferably below pH 8.4, more preferably below pH 8.3, further preferably below pH 8.2, and further preferably below pH 8.1. If adjusted to such a pH value, the genotype of the fimA gene of the Pg bacteria can be more accurately determined.

[0161] It is preferred that the liquid sample to be measured for fluorescence is subjected to constant temperature control adjusted to a specified temperature. The temperature of the liquid sample is preferably above 4°C and below 45°C. When a bacterial extract is used, the temperature of the liquid sample is preferably above 25°C, more preferably above 30°C, further preferably above 34°C, and further preferably above 36°C. In addition, it is preferably below 40°C, more preferably below 39°C, and further preferably below 38°C. On the other hand, when bacteria are used, the temperature of the liquid sample is preferably above 4°C, more preferably above 10°C, further preferably above 15°C, further preferably above 18°C, and further preferably above 21°C. In addition, it is preferably below 37°C, more preferably below 30°C, further preferably below 26°C, and further preferably below 23°C. If controlled to such a temperature, the genotype of the fimA gene of the Pg bacteria can be accurately determined.

[0162] Specifically, the comparison of the fluorescence measurement results in the sample group can be performed by comparing the fluorescence intensity value or the time-varying amount of fluorescence intensity of the liquid sample containing the pathogenic bacteria of periodontal disease or the bacterial extract (the test object) of unknown genotype in the sample group with the obtained measurement value group of the fluorescence intensity value or the time-varying amount of fluorescence intensity of the plurality of liquid samples in the sample group. When making the comparison, the fluorescence intensity values ​​or the time-varying amounts of fluorescence intensity are compared with each other.

[0163] When the bacterial cells are used to prepare the liquid sample, as a result of comparing the fluorescence measurement results (measurement value groups) in the sample group, when the fluorescence intensity value or the time-varying amount of fluorescence intensity obtained by measuring the liquid sample of the discrimination target of unknown genotype in the sample group is classified as the measurement value group ranked first among the measurement value groups of the fluorescence intensity values ​​or the time-varying amount of fluorescence intensity obtained by measuring the plurality of liquid samples in the sample group, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is type II. On the other hand, when the bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample classified as the measurement value group ranked first is type I.

[0164] like Figure 8 The shadow of the midpoint, Fig. 9 As shown by the shadow of the middle spot, the measurement value group ranked first in the front is the measurement value group in which the maximum value of the fluorescence intensity in all genotypes is classified. Similarly, regarding the time-dependent change (slope) of the fluorescence intensity, the measurement value group ranked first in the front is also the measurement value group in which the maximum value is classified. Therefore, when the bacterial cells are used to prepare the liquid sample and the sample group contains type II and type I or type IV, the measurement value group ranked first in the front is type II. On the other hand, when the bacterial cell extract is used to prepare the liquid sample and the sample group contains type I and type IV or type II, the measurement value group ranked first in the front is type I.

[0165] In addition, when bacterial cells are used to prepare liquid samples, or when bacterial cell extracts are used to prepare liquid samples, the results of comparison with the fluorescence measurement results (measurement value group) in the sample group are such that when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object whose genotype is unknown in the sample group is classified as the second-to-first measurement value group of the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring multiple liquid samples in the sample group, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is type IV.

[0166] like Figure 8 , Fig. 9As shown by the shadow of the middle oblique line, the second-ranked measurement value group is a measurement value group below the measurement value group classified only at the maximum value of the fluorescence intensity among all genotypes. Similarly, regarding the time-dependent change (slope) of the fluorescence intensity, the second-ranked measurement value group is also a measurement value group below the measurement value group classified only at the maximum value. Therefore, when the bacterial cells are used to prepare the liquid sample, and the sample group includes type II and type IV or type I, the second-ranked measurement value group is type IV. In addition, when the bacterial cell extract is used to prepare the liquid sample, and the sample group includes type I and type IV or type II, the second-ranked measurement value group is type IV.

[0167] Furthermore, when bacterial cells are used to prepare a liquid sample, as a result of comparison with the fluorescence measurement results (measurement value group) in the sample group, when the fluorescence intensity value or the time-varying amount of fluorescence intensity obtained by measuring the liquid sample of the discrimination target whose genotype is unknown in the sample group is classified as the last measurement value group among the measurement value groups of the fluorescence intensity values ​​or the time-varying amount of fluorescence intensity obtained by measuring a plurality of liquid samples in the sample group, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease (test bacteria) in the liquid sample is type I. On the other hand, when a bacterial cell extract is used to prepare a liquid sample, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample classified as the last measurement value group is type II.

[0168] like Figure 8 The shadow of the middle spot, Fig. 9 As shown by the shadow of the midpoint, the last measurement value group is the measurement value group classified by the minimum value of fluorescence intensity among all genotypes. Similarly, regarding the time-dependent change (slope) of fluorescence intensity, the last measurement value group is also the measurement value group classified by the minimum value. Therefore, when the bacterial cells are used to prepare the liquid sample and the sample group contains type I and type II or type IV, the last measurement value group is type I. On the other hand, when the bacterial cell extract is used to prepare the liquid sample and the sample group contains type II and type IV or type I, the last measurement value group is type II.

[0169] Comparison of the fluorescence measurement results in the sample group can be performed, for example, based on the similarity between the results obtained by the measurement. For example, the measurement value obtained by measuring the test area is compared with the measurement value obtained by measuring the control area. When the measurement value of the test area is within a predetermined range of similarity with respect to the measurement value of the control area, the fluorescence measurement results can be evaluated as being similar to each other.

[0170] The measured value group of the control area can be compared with the measured value of the test area in a state of being classified (clustered) by genotype in advance. Classification by genotype can use various calculation methods such as a method of classifying the measured value of each genotype according to a predetermined threshold, Ward's method, class average method, longest distance method, shortest distance method, etc. Comparison of fluorescence measurement results can be performed using various mathematical distances such as Euclidean distance.

[0171] According to the discrimination method involved in the above present embodiment, when there is a correlation between the enzyme activity of the pathogenic bacteria of periodontal disease and the genotype of the pathogenic bacteria of periodontal disease, the genotype of the pathogenic bacteria of periodontal disease can be discriminated according to the enzyme activity obtained by fluorescence determination. Different from the conventional molecular biological method, since the enzyme activity that makes the periodontal disease progress is reflected in the discrimination, it is possible to discriminate the genotype of the pathology more realistically. In addition, when the thalli are used to prepare a liquid sample, the sample collected from the oral cavity of the subject can be directly used for the liquid sample for fluorescence determination, so the genotype can be easily discriminated.

[0172] In particular, when the bacterial cells are used to prepare a liquid sample, if the pH is adjusted to be greater than 7.5 and less than pH 8.5 before the fluorescence measurement to allow the enzyme to react, the fluorescence intensity of type II or type IV becomes higher, thereby improving the accuracy of discrimination with genotypes other than type II or type IV. In addition, when the bacterial cell extract is used to prepare a liquid sample, if the pH is adjusted to be greater than 7.5 and less than pH 8.0 before the fluorescence measurement to allow the enzyme to react, the fluorescence intensity of type IV or type I becomes higher, thereby improving the accuracy of discrimination with genotypes other than type IV or type I. The test agent for discriminating the genotype of the pathogenic bacteria of periodontal disease can be adjusted to such a pH value in advance. The retention rate of type I Pg bacteria is high in adults with healthy periodontal tissues, while the retention rate of type II or type IV Pg bacteria is high in adult periodontitis patients, thereby being able to more accurately judge the current pathology of periodontal disease and the possibility of aggravation.

[0173] In addition, any of the bacterial cells contained in dental plaque, gum leaching fluid, etc. and the bacterial cell extracts extracted from the bacterial cells can also be used to prepare the liquid sample. It can be confirmed that the behavior of enzyme activity relative to temperature conditions shows a tendency to differ according to the genotype and the morphology of the object to be tested. The vesicles (vesicles) encapsulating enzymes and the like in the bacteria may differ from the freeness of the bacteria or the retention of the bacteria to them according to the genotype. However, by adjusting the conditions of the enzyme reaction according to the morphology of the object to be tested, high-precision discrimination can be performed.

[0174] <Fluorescence Measurement Device>

[0175] Next, refer to the attached Figure 1A fluorescence measuring device according to an embodiment of the present invention will be described.

[0176] Fig.10 It is a diagram showing the structure of a fluorescence measuring device according to an embodiment of the present invention.

[0177] like Fig.10 As shown, the fluorescence measuring device 100 involved in this embodiment has a light source (irradiation mechanism) 1, a sample rack 2, optical lenses 3a, 3b, a filter 4, a detection element (detection mechanism) 5, an amplifier 6, an analog processor 7, an A / D converter 8, a control device (discrimination mechanism) 9, a sample container 10, an input mechanism 11, a display mechanism 12, a pH measuring mechanism 13, a temperature measuring mechanism 14 and a temperature control device 15.

[0178] The fluorescence measuring device 100 involved in this embodiment is a fluorescence measuring device that can distinguish the genotype of the pathogen of periodontal disease. In the fluorescence measuring device 100, it is determined which genotype the pathogen of periodontal disease or its bacterial extract (test object) contained in the liquid sample Sa belongs to in the known polymorphic gene. The genotype is distinguished based on the enzyme activity that has been confirmed to be correlated with each genotype. The enzyme activity can be evaluated by using a test agent and a fluorescence measurement method, wherein the test agent uses a substrate for the enzyme reaction after fluorescence labeling.

[0179] As the pathogenic bacteria (test bacteria) of periodontal disease that are the objects of genotype discrimination, Porphyromonas gingivalis (Pg bacteria) can be listed in the same manner as the discrimination method involved in the above-mentioned embodiment. As the genotype to be discriminated, type I (type 1), type II (type 2), type III (type 3), type IV (type 4), and type V (type 5) of the polymorphic fimA gene can be listed.

[0180] As the bacteria to be tested, similarly to the discrimination method involved in the above-mentioned embodiment, dental plaque, gingival extract, etc. collected from the oral cavity of the subject can be directly used, or it can also be used as a suspension or a supernatant of the suspension. As a liquid sample for fluorescence measurement, a liquid sample Sa can be used, which contains a periodontal disease pathogen or its bacterial extract and a fluorescently labeled substrate (reagent) formed by an enzyme reaction substrate of the fluorescently labeled periodontal disease pathogen.

[0181] The light source 1 is a device for generating excitation light. When the pathogenic bacteria of periodontal disease cause an enzyme reaction in the liquid sample Sa, the fluorescent chromophore dissociates from the fluorescent labeling substrate. When the excitation light generated by the light source 1 is irradiated to the liquid sample Sa, the fluorescent light emitted by the fluorescent chromophore is emitted from the liquid sample Sa.

[0182] As the light source 1, a monochromatic light source with a specific excitation wavelength is preferably used, such as a light emitting diode (LED), a laser light source, etc. However, as the light source 1, other light sources such as a xenon lamp, a mercury lamp, a halogen lamp, etc. may be used together with an optical system such as a spectrometer.

[0183] The excitation light emitted by the light source 1 preferably has a maximum peak at a wavelength of 350 nm to 380 nm, more preferably has a maximum peak at a wavelength of 355 nm to 375 nm, and further preferably has a maximum peak at a wavelength of 360 nm to 370 nm. If such a spectrum is obtained, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, a fluorescence intensity suitable for detection can be obtained.

[0184] The sample rack 2 is installed in a measurement chamber (not shown) and supports the sample container 10 so that it can emit excitation light and fluorescence. When performing fluorescence measurement, the sample container 10 containing the liquid sample Sa is fixed to the sample rack 2. The sample container 10 supported by the sample rack 2 is irradiated with excitation light from the side.

[0185] The detection element 5 is a device for detecting the fluorescence emitted by the liquid sample Sa. The fluorescence emitted from the liquid sample Sa is emitted from the sample container 10 and reaches the filter 4 through the optical lens 3a. The filter 4 removes noise and low-sensitivity bands and only allows the fluorescence of a specific band to pass. The fluorescence after passing through the filter 4 is incident on the detection element 5 through the optical lens 3b and is converted into an electrical signal.

[0186] Various detection elements such as photodiodes, phototubes, and photomultiplier tubes can be used as detection element 5. Optical filters, dichroic mirrors, and the like can be used as filter 4. It is preferred that a filter that transmits wavelengths of 410 nm to 475 nm and shields wavelengths other than these is used as filter 4. With such characteristics, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, fluorescence can be detected with high sensitivity.

[0187] The fluorescent electrical signal converted by the detection element 5 is amplified by the amplifier 6 and then subjected to noise removal processing by the analog processor 7 having a low-pass filter etc. Thereafter, the fluorescent electrical signal is converted into a digital signal by the A / D converter 8 and input to the control device 9.

[0188] Preferably, before the fluorescence measurement, the liquid sample Sa of the fluorescence measurement object is adjusted to a pH greater than 7.5 and below pH 8.5 to allow the enzyme reaction to proceed. The pH value of the liquid sample is preferably above pH 7.8. In addition, the pH value of the liquid sample is preferably below pH 8.4, more preferably below pH 8.3, further preferably below pH 8.2, and further preferably below pH 8.1. If adjusted to such a pH value, the genotype of the fimA gene of the Pg bacteria can be more accurately determined.

[0189] The pH value of the liquid sample Sa can be measured by the pH measuring mechanism 13. For example, a glass electrode type, membrane electrode type, or other pH meter can be inserted into the sample container 10 and used as the pH measuring mechanism 13. According to the pH measuring mechanism 13, the pH value of the liquid sample Sa can be measured when performing fluorescence measurement, so when the pH value is not within a predetermined range, it is possible to deal with it, either by stopping the fluorescence measurement, or discarding an inaccurate fluorescence measurement result.

[0190] The liquid sample Sa of the fluorescence measurement object is preferably subjected to fluorescence measurement under thermostatic control adjusted to a specified temperature. The temperature of the liquid sample is preferably above 4°C and below 45°C. When a bacterial extract is used, the temperature of the liquid sample is preferably above 25°C, more preferably above 30°C, further preferably above 34°C, and further preferably above 36°C. In addition, it is preferably below 40°C, more preferably below 39°C, and further preferably below 38°C. On the other hand, when bacterial cells are used, the temperature of the liquid sample is preferably above 4°C, more preferably above 10°C, further preferably above 15°C, further preferably above 18°C, and further preferably above 21°C. In addition, it is preferably below 37°C, more preferably below 30°C, further preferably below 26°C, and further preferably below 23°C. If controlled to such a temperature, the genotype of the fimA gene of the Pg bacteria can be accurately determined.

[0191] The temperature of the liquid sample Sa can be measured by the temperature measuring mechanism 14. For example, a thermistor, a thermocouple, a temperature measuring resistor, etc. can be inserted into the sample container 10 and used as the temperature measuring mechanism 14. According to the temperature measuring mechanism 14, when performing fluorescence measurement, the temperature of the liquid sample Sa can be monitored online, and the temperature control device 15 can be feedback controlled.

[0192] The temperature control device 15 is a device for controlling the temperature of the liquid sample Sa in the sample container 10. A PTC (Positive Temperature Coefficient) heater, a Peltier element, a constant temperature medium circulation system, etc. can be provided around the sample container 10, for example, on the sample rack 2 below or on the side thereof as the temperature control device 15. According to the temperature control device 15, the temperature of the liquid sample Sa can be controlled to a temperature suitable for the enzyme reaction, thereby accurately evaluating the enzyme activity.

[0193] In addition, Fig.10 In the identification device 100 shown, a grid-shaped sample container 10 is used, but a microtube may be used as the sample container 10. Examples of the microtube include containers made of plastic or glass with a volume of about 1 to 2 mL that can be used for various operations such as reaction, extraction, culture, and centrifugation.

[0194] When a microtube is used as the sample container 10, the microtube containing the liquid sample Sa in an open state can be mounted on the sample rack. The excitation light can be injected into such a microtube from the side wall surface. The fluorescence emitted from the liquid sample Sa can be emitted to the upper side of the microtube in the open state for detection, rather than being emitted to the side of the sample rack.

[0195] According to this method, the side wall of the microtube becomes an optical waveguide for the excitation light, and the fluorescence is emitted toward the top of the microtube in the open state, so the optical system can be simplified. In addition, the microtube used for various operations can be directly used for fluorescence measurement. Therefore, a small amount of liquid sample Sa can be used as the measurement object, and fluorescence measurement can be performed cheaply and simply using a fluorescence measurement device with a simple structure.

[0196] Fig.11 This is a diagram showing a schematic configuration of a control device included in the fluorescence measuring device.

[0197] like Fig.11 As shown, the control device 9 of the fluorescence measurement device 100 includes a measurement result data acquisition unit 90, a measurement result data processing unit 91, a measurement result data comparison unit 92, a measurement condition data acquisition unit 93, a control unit 94, a storage unit 95, a display control unit 96 and a temperature control unit 97.

[0198] The control device 9 controls the operation of the fluorescence measuring device 100 and performs processing for distinguishing the genotype of the pathogenic bacteria and bacterial extract of periodontal disease contained in the liquid sample Sa. The control device 9 can be composed of a computing device such as a CPU (Central Processing Unit), a ROM (Read Only Memory), a RAM (Random Access Memory), a hard disk and other storage devices.

[0199] The control device 9 is connected to an input mechanism 11, a fluorescence measuring unit having a detection element 5, a pH measuring mechanism 14, and a temperature measuring mechanism 15 via an input interface not shown. In addition, the control device 9 is connected to a display mechanism 12 and a temperature control device 15 via an output interface not shown. The various components of the control device 9 are connected to each other via a bus not shown.

[0200] The input mechanism 11 is a device for operating the fluorescence measurement device 100. The input mechanism 11 can be constituted by various devices such as a keyboard, a mouse, and a touch panel.

[0201] The display unit 12 is a device for displaying the result of genotype discrimination. The display unit 12 can be composed of various devices such as a liquid crystal display, a plasma display, and an organic EL display.

[0202] In the fluorescence measuring device 100, the genotype of the periodontal disease pathogen or bacterial extract contained in the liquid sample Sa can be determined by comparing the fluorescence intensity value or the time-varying amount of the fluorescence intensity of the liquid sample containing the periodontal disease pathogen or bacterial extract (test object) of unknown genotype with a preset threshold value. When making the comparison, the fluorescence intensity value is compared with the threshold value corresponding to the fluorescence intensity value, or the time-varying amount of the fluorescence intensity is compared with the threshold value corresponding to the time-varying amount of the fluorescence intensity.

[0203] The measurement result data acquisition unit 90 acquires the measurement result data input from the fluorescence measurement unit having the detection element 5. The measurement result data is data such as the fluorescence intensity of the fluorescence obtained by detecting the liquid sample Sa, the detection time calculated from the start time of the enzyme reaction, etc. The measurement result data is output to the measurement result data processing unit 91 and the storage unit 95.

[0204] The measurement result data processing unit 91 calculates the fluorescence intensity data for distinguishing the genotype based on the measurement result data. The fluorescence intensity data is data such as the fluorescence intensity value of the specified detection time, the time-varying amount (slope) of the fluorescence intensity of the specified detection time, etc. The fluorescence intensity data can also be the average value of the fluorescence intensity value within the specified detection time range, the average value or maximum value of the time-varying amount (slope) of the fluorescence intensity within the specified detection time range, etc. The fluorescence intensity data is output to the measurement result data comparison unit 92 and the storage unit 95.

[0205] The measurement result data comparison unit 92 compares the fluorescence intensity data with the threshold value stored in the storage unit 95. The measurement result data comparison unit 92 determines whether the fluorescence intensity data generated for the liquid sample Sa is greater than the threshold value, thereby discriminating the genotype of the pathogenic bacteria or bacterial extract of periodontal disease contained in the liquid sample Sa. Data indicating the discrimination result is output to the display control unit 97.

[0206] The measurement condition data acquisition unit 93 acquires the measurement condition data input from the pH measurement unit 13 and the temperature measurement unit 14. The measurement condition data is data on the pH value of the liquid sample Sa measured at a predetermined time interval by the pH measurement unit 13 and the temperature of the liquid sample Sa measured at a predetermined time interval by the temperature measurement unit 14. The measurement condition data is output to the control unit 95 and the temperature control unit 97.

[0207] The control unit 94 controls the operation of each device included in the fluorescence measurement device 100 , the process of distinguishing the genotype of the pathogenic bacteria of periodontal disease, the process of displaying the distinction result, etc., based on a predetermined program and the input of the user via the input unit 11 .

[0208] The storage unit 95 stores programs for executing the operation of each device included in the fluorescence measurement device 100 , processing for distinguishing the genotype of periodontal disease pathogens, processing for displaying the distinction results, and data such as threshold values ​​for distinguishing genotypes.

[0209] For example, fluorescence measurement can be performed in advance using pathogenic bacteria or bacterial extracts of periodontal disease whose genotypes are known, and fluorescence intensity data can be obtained based on the measurement result data obtained as the measurement results, and the data can be pre-stored in the storage unit 95. In addition, a threshold value for each genotype can be set based on a plurality of fluorescence intensity data, and the data can be pre-stored in the storage unit 95.

[0210] The threshold value can be set using various analysis methods such as regression analysis, standard deviation classification, natural classification, and multinomial classification. The threshold value may be calibrated in advance so as not to be affected by enzyme activities other than the degrading enzyme to be evaluated.

[0211] The display control unit 96 controls the generation and display of images to be displayed on the display unit 12 . The display control unit 96 generates images regarding the operating state of the fluorescence measurement device 100 , the results of fluorescence measurement, and the results of genotype discrimination, and outputs them to the display unit 12 .

[0212] The temperature control unit 97 controls the temperature of the temperature control device 15. The temperature control unit 97 performs feedback control on the temperature control device 15 based on the temperature of the liquid sample Sa measured by the temperature measuring mechanism 15, thereby maintaining the temperature of the liquid sample Sa at a temperature suitable for the enzyme reaction.

[0213] Fig.12 This is a flowchart showing the flow of a discrimination method implemented by the fluorescence measuring device.

[0214] like Fig.12 As shown, in the fluorescence measurement device 100, a liquid sample Sa containing periodontal disease pathogens or their bacterial extracts (test objects) with unknown genotypes can be used as the measurement object for fluorescence measurement, and the genotype discrimination results analyzed based on the measurement data can be displayed to the user.

[0215] like Fig.12 As shown, when the fluorescence measurement device 100 is operated, first, the measurement conditions of the fluorescence measurement are input to the control device 9 (step S300). Examples of the measurement conditions include the type of the sample to be measured for preparing the liquid sample, the fluorescence wavelength for fluorescence measurement, the standby time, the detection time, and the type of fluorescence intensity data for distinguishing genotypes, such as the fluorescence intensity value at a predetermined detection time, the time-dependent change amount (slope) of the fluorescence intensity at a predetermined detection time, and the like.

[0216] Next, the fluorescence measurement of the liquid sample Sa containing the periodontal disease pathogen of unknown genotype or its bacterial extract (test object) is started, and the excitation light is irradiated and the fluorescence is detected (step S310). Then, the measurement result data of the electrical signal as fluorescence detected by the detection element 5 is obtained (step S320).

[0217] In step S320, when the fluorescence intensity value at a predetermined detection time is used to distinguish the genotype, the fluorescence intensity value at that time is collected as the measurement result data in the measurement result data acquisition unit 90. In addition, when the time-dependent change amount (slope) of the fluorescence intensity is used to distinguish the genotype, the fluorescence intensity value at a predetermined time interval and over time is collected as the measurement result data in the measurement result data acquisition unit 90. In addition, when the average value or the maximum value is used, the fluorescence intensity value of the predetermined time range is collected.

[0218] Next, the fluorescence intensity data for distinguishing the genotype is calculated based on the measurement result data (step S330). The fluorescence intensity data is collected in the measurement result data processing unit 91 as the fluorescence intensity value at the specified detection time, the average value of the fluorescence intensity value within the specified detection time range, the time change amount (slope) of the fluorescence intensity at the specified detection time, the average value or maximum value of the time change amount (slope) of the fluorescence intensity within the specified detection time range, etc.

[0219] Next, the genotype of the periodontal disease pathogen or its bacterial extract (test object) with unknown genotype is determined (step S340). Then, the display control unit 96 displays an image showing the genotype determination result on the display mechanism 12 (step S350). After that, the operation of the fluorescence measurement device 100 is terminated.

[0220] As the result of genotype discrimination, the display unit 12 can display whether the periodontal disease pathogen or bacterial extract of unknown genotype contained in the liquid sample Sa belongs to a known genotype, does not belong to a known genotype, cannot be discriminated, etc. The discrimination result can be displayed in any form such as language, symbol, color, etc., and can also be displayed together with a percentage indicating the discrimination accuracy, etc.

[0221] Fig.13 This is a flowchart showing the flow of genotype discrimination processing performed by the fluorescence measurement device.

[0222] like Fig.13 As shown, the process of distinguishing the genotype of the pathogen of periodontal disease or the bacterial extract with unknown genotype (step S340) is performed by comparing the fluorescence intensity data, which is the fluorescence intensity value or the time-dependent change of the fluorescence intensity, with the threshold value stored in the storage unit 95 in the measurement result data comparison unit 92.

[0223] First, the fluorescence intensity data obtained for a liquid sample containing periodontal disease pathogenic bacteria or a bacterial cell extract thereof (analyte) of unknown genotype is input to the measurement result data comparison unit 92 (step S341 ).

[0224] Next, the fluorescence intensity data of the liquid sample containing periodontal disease pathogens of unknown genotype or their bacterial extracts (analytes), that is, the fluorescence intensity value or the amount of change in fluorescence intensity over time, is compared with a preset first threshold value (step S342).

[0225] As a result of comparison with the first threshold, if the fluorescence intensity data of the liquid sample with unknown genotype, that is, the fluorescence intensity value or the amount of change in fluorescence intensity over time, is greater than the first threshold (step S342 : Yes), the process proceeds to step S343 .

[0226] Next, the fluorescence intensity data of the liquid sample containing periodontal disease pathogens (test bacteria) of unknown genotype, that is, the fluorescence intensity value or the amount of change in fluorescence intensity over time is compared with a preset second threshold value (step S343).

[0227] The first threshold or the second threshold can be set as follows: using periodontal disease pathogens or bacterial extracts with known genotypes to perform fluorescence measurement in advance, and setting an arbitrary value based on the correlation between fluorescence intensity and reaction time according to the genotype of the identification object and the conditions of the enzyme reaction as the first threshold or the second threshold. Figure 8 or Fig. 9 The shaded areas in the figure are divided into the boundary values ​​that distinguish between type I and type IV, the boundary values ​​that distinguish between type IV and type II, and the like.

[0228] In the case where the bacterial cells are used to prepare the liquid sample, for example, the first threshold value can be set based on at least one of the fluorescence intensity value or the time-varying amount of the fluorescence intensity of the liquid sample containing the bacterial cells of the pathogenic bacteria of the periodontal disease of the genotype I of the fimA gene, and the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample containing the bacterial cells of the pathogenic bacteria of the genotype IV of the fimA gene. For example, a plurality of fluorescence measurement results can be obtained in advance for the liquid sample having substantially the same conditions as the liquid sample Sa of the discrimination object, such as pH value, temperature, the amount of the pathogenic bacteria to be measured, and the concentration of the fluorescent labeled substrate, and a boundary value can be set based on the result of the time-varying amount of the fluorescence intensity, and the boundary value is less than the maximum value of the fluorescence measurement results collected for type I and less than the minimum value of the fluorescence measurement results collected for type IV. In the case where the bacterial cells are used to prepare the liquid sample, type I can be distinguished from type IV or type II based on the first threshold value.

[0229] On the other hand, when a bacterial extract is used to prepare a liquid sample, the first threshold value can be set based on at least one of a fluorescence intensity value or a time-varying amount of fluorescence intensity of a liquid sample of a bacterial extract of a pathogenic bacterium of periodontal disease containing a genotype II of the fimA gene, and a fluorescence intensity value or a time-varying amount of fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogenic bacterium containing a genotype IV of the fimA gene. When a bacterial extract is used to prepare a liquid sample, type II can be distinguished from type IV or type I based on the first threshold value.

[0230] In the case where the bacterial cells are used to prepare the liquid sample, the second threshold value can be set based on at least one of the fluorescence intensity value or the time-varying amount of the fluorescence intensity of the liquid sample containing the bacterial cells of the pathogenic bacteria of the periodontal disease of type IV genotype of the fimA gene, and the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample containing the bacterial cells of the pathogenic bacteria of type II genotype of the fimA gene. For example, a plurality of fluorescence measurement results can be obtained in advance for a liquid sample having substantially the same conditions as the liquid sample Sa of the discrimination object, such as pH value, temperature, the amount of the pathogenic bacteria to be measured, and the concentration of the fluorescent labeled substrate, and a boundary value can be set based on the result of the time-varying amount of the fluorescence intensity, and the boundary value is less than the maximum value of the fluorescence measurement results collected for type IV and less than the minimum value of the fluorescence measurement results collected for type II. In the case where the bacterial cells are used to prepare the liquid sample, type II can be distinguished from type IV or type I based on the second threshold value.

[0231] On the other hand, when the bacterial extract is used to prepare the liquid sample, the second threshold value can be set based on at least one of the fluorescence intensity value or the amount of change in fluorescence intensity over time of the liquid sample of the bacterial extract of the pathogenic bacteria of periodontal disease whose genotype is type IV of the fimA gene, and the fluorescence intensity value or the amount of change in fluorescence intensity over time obtained by measuring the liquid sample of the bacterial extract of the pathogenic bacteria whose genotype is type I of the fimA gene. When the bacterial extract is used to prepare the liquid sample, type I can be distinguished from type IV or type II based on the second threshold value.

[0232] In the case where the bacterial cells are used to prepare the liquid sample, the result of comparison with the threshold value is that when the fluorescence intensity data of the liquid sample with unknown genotype, that is, the fluorescence intensity value or the time-dependent change of the fluorescence intensity is greater than the first threshold value and is less than the second threshold value (step S342: yes, step S343: no), it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type I and type II. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type IV. In addition, in the case where the bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type II and type I. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type IV.

[0233] In addition, when the bacterial cells are used to prepare the liquid sample, the result of comparison with the threshold value is that when the fluorescence intensity data of the liquid sample with unknown genotype, that is, the fluorescence intensity value or the time-dependent change of the fluorescence intensity is greater than the second threshold value (step S342: yes, step S343: yes), it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type I and type IV. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type II. In addition, when the bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type II and type IV. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type I.

[0234] On the other hand, when the bacterial cells are used to prepare the liquid sample, as a result of comparison with the threshold value, when the fluorescence intensity data of the liquid sample of unknown genotype, that is, the fluorescence intensity value or the time-dependent change of the fluorescence intensity is below the first threshold value (step S342: No), it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type II and type IV. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type I. In addition, when the bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the pathogenic bacteria of periodontal disease in the liquid sample is a genotype other than type IV and type I. That is, if the bacteria to be tested do not contain genotypes other than type III and type V, it can be determined that it is type II.

[0235] According to the fluorescence measuring device involved in the above embodiment, when there is a correlation between the enzyme activity of the pathogen of periodontal disease and the genotype of the pathogen of periodontal disease, the genotype of the pathogen of periodontal disease can be distinguished according to the enzyme activity obtained by fluorescence measurement. Unlike the conventional molecular biological method, since the enzyme activity that causes the progression of periodontal disease is reflected in the discrimination, a more practical discrimination of the genotype of the pathology can be performed. In addition, the sample collected from the oral cavity of the subject can be directly used as a liquid sample for fluorescence measurement, so the genotype can be easily distinguished.

[0236] In particular, the fluorescence measuring device can include a storage unit storing the first threshold value and the second threshold value, so that the genotype of the pathogenic bacteria of periodontal disease can be stably and reproducibly determined regardless of the skill and operation. By preparing only a test agent for determining the genotype of the pathogenic bacteria of periodontal disease, the sample collected from the oral cavity of a test subject of the pathogenic bacteria of periodontal disease can be automatically determined, so the current pathology of periodontal disease and the possibility of aggravation can be determined efficiently.

[0237] The present invention is described above, but the present invention is not limited to the above embodiments, and various changes can be made within the scope of the present invention. For example, the present invention is not necessarily limited to having all the structures of the above embodiments. A part of the structure of a certain embodiment can be replaced with another structure, or another method can be added to a part of the structure of the embodiment, or a part of the structure of a certain embodiment can be omitted.

[0238] For example, the fluorescence measuring device 100 can be provided with an appropriate optical system and a signal processing system as long as it can measure the fluorescence intensity of the liquid sample Sa. In addition to the first threshold value or the second threshold value, the genotype can be distinguished by other methods such as comparing the similarity of the index value of the fluorescence intensity, as in the same method as the above-mentioned distinguishing method.

[0239] [Explanation of Reference Numerals]

[0240] 1: light source (irradiation mechanism); 2: sample rack; 3a: optical lens; 3b: optical lens; 4: filter; 5: detection element (detection mechanism); 6: amplifier; 7: analog processor; 8: A / D converter; 9: control device (discrimination mechanism); 10: sample container; 11: input mechanism; 12: display mechanism; 13: pH measurement mechanism; 14: temperature measurement mechanism; 15: temperature control device; 90: measurement result data acquisition unit; 91: measurement result data processing unit; 92: measurement result data comparison unit; 93: measurement condition data acquisition unit; 94: control unit; 95: storage unit; 96: display control unit; 97: temperature control unit; 100: fluorescence measurement device.

Claims

1. A fluorescence measuring device for distinguishing the genotype of pathogenic bacteria of periodontal disease, characterized in that: The pathogen is Porphyromonas gingivalis. The genotype includes type I, type II and type IV, which are genotypes of the fimA gene encoding the fimbrial protein. It has an irradiation mechanism, a detection mechanism and a discrimination mechanism, among which: The irradiation mechanism is used to irradiate the liquid sample with excitation light, the liquid sample contains bacteria or bacterial extracts of periodontal disease pathogens, and a fluorescently labeled reagent that is a substrate for an enzyme reaction with the pathogens, the reagent is a polypeptide whose N-terminus is protected by a protecting group, i.e., protecting group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide, and is adjusted to a pH of not less than 7.0 and not more than 8.5 for enzyme reaction, and a temperature of not less than 4° C. and not more than 45° C.; The detection mechanism is used to detect the fluorescence emitted from the liquid sample; The discrimination means discriminates the genotype based on the intensity of the fluorescence detected by the detection means, The determination mechanism comprises a storage unit and a data comparison unit, wherein: The storage unit is used to store the fluorescence intensity value or the time-dependent change of the fluorescence intensity obtained by measuring the control area. The storage unit of the determination means stores a first threshold value and a second threshold value, (1) when the liquid sample selects the bacterial body of the pathogen, the first threshold value is a value preset based on at least one of a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the bacterial body of the pathogen of genotype I containing the fimA gene, and a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the bacterial body of the pathogen of genotype IV containing the fimA gene, The second threshold value is a value preset based on at least one of a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the pathogenic bacteria of genotype IV containing the fimA gene, and a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the pathogenic bacteria of genotype II containing the fimA gene, The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the first threshold value, and when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is below the first threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type I, The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the first threshold value and the second threshold value set in advance, and when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is greater than the first threshold value and is less than the second threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type IV, The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the preset second threshold value, and when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is greater than the second threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type II, (2) when the liquid sample is a bacterial extract of the pathogen, the first threshold value is a value pre-set based on at least one of a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogen having a genotype II containing the fimA gene, and a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogen having a genotype IV containing the fimA gene, The second threshold value is a value preset based on at least one of a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogenic bacteria of genotype IV containing the fimA gene, and a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogenic bacteria of genotype I containing the fimA gene, The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the first threshold value, and when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is below the first threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type II, The data comparison unit compares the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the first threshold value and the second threshold value set in advance. When the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample of the discrimination object is greater than the first threshold value and is less than the second threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type IV. The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the identification object with the pre-set second threshold value. When the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the identification object is greater than the second threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type I.

2. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample of a test area containing bacterial cells or bacterial cell extracts of the pathogenic bacteria whose genotype is unknown, and a liquid sample in the control zone containing the bacterial cells or bacterial extract of the pathogenic bacteria of known genotype and adjusted to the same pH value as the liquid sample in the test zone, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the test area is the same as or similar to the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the control area, it is determined that the fimA gene of the pathogen in the liquid sample in the test area is the same genotype as the fimA gene of the pathogen whose genotype is known.

3. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains bacterial cells of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing bacterial cells of the pathogenic bacteria with known genotypes or bacterial cells of the pathogenic bacteria with unknown genotypes and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group in which the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, the fimA gene of the pathogen of the liquid sample is judged to be type II.

4. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains bacterial cells of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing bacterial cells of the pathogenic bacteria with known genotypes or bacterial cells of the pathogenic bacteria with unknown genotypes and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group of the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, the fimA gene of the pathogen of the liquid sample is judged to be type IV.

5. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains bacterial cells of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing bacterial cells of the pathogenic bacteria with known genotypes or bacterial cells of the pathogenic bacteria with unknown genotypes and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group of the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, when the measurement value group is ranked last, it is judged that the fimA gene of the pathogen of the liquid sample is type I.

6. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains a bacterial extract of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing a bacterial extract of the pathogen with a known genotype or a bacterial extract of the pathogen with an unknown genotype and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group in which the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, the fimA gene of the pathogen of the liquid sample is judged to be type I.

7. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains a bacterial extract of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing a bacterial extract of the pathogen with a known genotype or a bacterial extract of the pathogen with an unknown genotype and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group of the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, the fimA gene of the pathogen of the liquid sample is judged to be type IV.

8. The fluorescence measuring device according to claim 1, characterized in that: The liquid sample is a liquid sample to be identified that contains a bacterial extract of the pathogenic bacteria whose genotype is unknown, and a plurality of liquid samples containing a bacterial extract of the pathogen with a known genotype or a bacterial extract of the pathogen with an unknown genotype and adjusted to the same pH value as the liquid sample to be identified, In the discrimination of the genotype, when the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the discrimination object is classified into a measurement value group of the fluorescence intensity values ​​or the time-dependent change in fluorescence intensity obtained by measuring the multiple liquid samples, and when the above-mentioned fluorescence intensity values ​​or the time-dependent change in fluorescence intensity are arranged in order from high to low, when the measurement value group is ranked last, it is judged that the fimA gene of the pathogen of the liquid sample is type II.

9. A fluorescence measuring device for determining the genotype of pathogenic bacteria of periodontal disease, characterized in that: The pathogen is Porphyromonas gingivalis. The genotype includes type I, type II, and type IV, which are genotypes of the fimA gene encoding the fimbrial protein. It has an irradiation mechanism, a detection mechanism and a discrimination mechanism, among which: The irradiation mechanism is used to irradiate the liquid sample with excitation light, the liquid sample contains bacteria or bacterial extracts of periodontal disease pathogens, and a fluorescently labeled reagent that is a substrate for an enzyme reaction with the pathogens, the reagent is a polypeptide whose N-terminus is protected by a protecting group, i.e., protecting group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide, and is adjusted to a pH of not less than 7.0 and not more than 8.5 for enzyme reaction, and a temperature of not less than 4° C. and not more than 45° C.; The detection mechanism is used to detect the fluorescence emitted from the liquid sample; The discrimination means discriminates the genotype based on the intensity of the fluorescence detected by the detection means, The determination mechanism comprises a storage unit and a data comparison unit, wherein: The storage unit is used to store the fluorescence intensity value or the time-dependent change of the fluorescence intensity obtained by measuring the control area. The storage unit of the determination means stores a threshold value. (1) when the liquid sample selects the bacterial body of the pathogen, the threshold value is a value preset based on at least one of the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the bacterial body of the pathogen whose genotype is IV containing the fimA gene, and the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the bacterial body of the pathogen whose genotype is II containing the fimA gene, The data comparison unit compares the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample of the discrimination object with the preset threshold value, and when the fluorescence intensity value or the time-varying amount of the fluorescence intensity obtained by measuring the liquid sample of the discrimination object is greater than the threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type II, (2) when the liquid sample is a bacterial extract of the pathogen, the threshold value is a value pre-set based on at least one of a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogen having a genotype II containing the fimA gene, and a fluorescence intensity value or a time-dependent change in fluorescence intensity obtained by measuring the liquid sample of a bacterial extract of the pathogen having a genotype IV containing the fimA gene, The data comparison unit compares the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the identification object with the threshold value. When the fluorescence intensity value or the time-dependent change in fluorescence intensity obtained by measuring the liquid sample of the identification object is below the threshold value, it is determined that the fimA gene of the pathogen in the liquid sample is type II.

10. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: A display device is provided, and the display device is used to display the discrimination result of the genotype.

11. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: A pH measuring mechanism is provided, and the pH measuring mechanism is used to measure the pH value of the liquid sample.

12. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: A temperature control mechanism is provided, and the temperature control mechanism is used to adjust the temperature of the liquid sample.

13. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: The reagent is isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA).

14. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: The wavelength of the excitation light is greater than or equal to 355 nm and less than or equal to 375 nm.

15. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: The wavelength of the fluorescence is greater than or equal to 430 nm and less than or equal to 455 nm.

16. The fluorescence measuring device according to any one of claims 1 to 9, characterized in that: The liquid sample comprises a pH buffer solution in which the reagent is dissolved, The pH buffer contains tris(hydroxymethyl)aminomethane (Tris) as a main component.

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