JP7479610B2 - Discrimination method and fluorescence measuring device - Google Patents

Description

The present invention relates to a method for identifying the genotype of bacteria that cause periodontal disease and a fluorescence measuring device.

There are several hundred types of oral bacteria in the human oral cavity. Among these oral bacteria, three species, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia, are classified as the Red complex, which is closely related to periodontal disease.

Periodontal disease is an inflammatory disease caused by oral bacteria, and not only does it affect periodontal tissues, but it has also been noted to be linked to systemic diseases such as myocardial infarction, diabetes, and arteriosclerosis. Traditionally, periodontal disease has been diagnosed using probing tests that use a probe to check the depth of periodontal pockets and the presence or absence of bleeding, as well as X-ray tests to observe the alveolar bone. In-vitro diagnostic test agents have also been developed to check for the presence of bacteria that cause periodontal disease.

In the fields of bacteriology and clinical practice, it is believed that of the three types of bacteria classified as the red complex, Porphyromonas gingivalis (Pg) is the one that has a significant impact on the aggravation of periodontal disease, i.e., the progression of alveolar bone resorption. Pg bacteria have pili on the surface of their body, and it is known that components of the bacterial surface, such as pili, are closely related to their attachment and colonization in the oral cavity.

The gene that codes for the fimbria protein of Pg bacteria is the fimA gene, which codes for a subunit of the fimbria protein. The fimA gene has been confirmed to show genetic polymorphism and is classified into five types, I to V (types 1 to 5). Previously, it has been reported that the ability of Pg bacteria to adhere and colonize in the oral cavity and its pathogenicity may differ depending on the genotype of the fimA gene.

For example, Non-Patent Document 1 reports that adults with healthy periodontal tissues had a rate of Type I of the fimA gene of approximately 70%, Type V of approximately 30%, and others of less than 10%, while adult periodontitis patients had a rate of Type II (Type 2) of approximately just under 60%, Type IV (Type 4) of approximately just under 20%, and others of less than 10%. It has been reported that more than 90% of Pg bacteria detected in patients with advanced periodontitis were Type II.

Atsuo Amano, "Relationship between the adhesion ability of Porphyromonas gingivalis fimbriae and periodontal pathogenicity of genetic polymorphisms," Journal of the Japanese Society of Periodontology, 2003, Vol. 45, No. 4, pp. 357-363

It has been suggested that the genotypes of bacteria that cause periodontal disease may have different effects on the pathology of periodontal disease, such as the fimA gene of Pg bacteria. Therefore, if it were possible to distinguish the genotypes that make up a patient's oral bacterial flora when diagnosing, treating, and preventing periodontal disease, it may be possible to appropriately evaluate the pathology of periodontal disease and predict the possibility of it becoming severe.

Conventionally, molecular biology techniques using RT-PCR and the like have been used to distinguish genotypes. However, molecular biology techniques require time-consuming operations and are difficult to carry out easily. Furthermore, while molecular biology techniques can indirectly measure the number of gene copies or the number of cells, they do not measure the enzyme activity that actually causes periodontal disease to progress.

Pg bacteria, classified as part of the red complex, produce gingipain, a type of protease, and it is believed that these enzymes cause disruption of the bacterial flora, such as dysbiosis, and promote inflammatory reactions, which in turn progress periodontal disease. Therefore, there is a demand for a method of distinguishing genotypes based on direct indicators that are easier to perform than conventional molecular biology methods and more in line with the pathology of periodontal disease.

The present invention aims to provide a method and a fluorescence measuring device that can easily distinguish the genotype of bacteria that cause periodontal disease based on enzyme activity.

In order to solve the above-mentioned problems, the discrimination method of the present invention is a discrimination method for discriminating the genotype of a causative bacterium of periodontal disease , wherein the causative bacterium is Porphyromonas gingivalis, and the genotype is a polymorphism of the fimA gene encoding a pilus protein, and the discrimination method includes: a bacterial cell extract of the causative bacterium collected from the oral cavity of a subject, or a supernatant collected by centrifuging a cell suspension of the causative bacterium; and a reagent in which a substrate for an enzyme reaction by the causative bacterium is fluorescently labeled , the reagent being a protecting group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide, which is a polypeptide whose N-terminus is protected by a protecting group; and the liquid sample subjected to an enzyme reaction with the liquid temperature adjusted to between 4°C and 38°C is irradiated with excitation light, and the genotype is discriminated based on the change over time in the fluorescence intensity of the fluorescence emitted from the liquid sample.

Furthermore, the fluorescence measuring device according to the present invention is a fluorescence measuring device for discriminating the genotype of a causative bacterium of periodontal disease , the causative bacterium being Porphyromonas gingivalis, the genotype being a polymorphism of the fimA gene encoding a pilus protein, the device comprising: cells of the causative bacterium collected from the oral cavity of a subject, or a cell extract of the causative bacterium which is a supernatant collected by centrifuging a cell suspension of the causative bacterium; and protecting group-glycyl- glycyl-L-arginyl-4-methylcoumaryl-7-amide which is a polypeptide whose N-terminus is protected by a protecting group as a reagent in which a substrate for an enzyme reaction by the causative bacterium is fluorescently labeled; and the device is equipped with an irradiation means for irradiating an excitation light onto a liquid sample which has been subjected to an enzyme reaction by adjusting the liquid temperature to 4°C or higher and 38°C or lower; a detection means for detecting the fluorescence emitted from the liquid sample; and a discrimination means for discriminating the genotype based on the amount of change over time in the fluorescence intensity of the detected fluorescence.

The present invention provides a method and a fluorescence measuring device that can easily distinguish the genotype of bacteria causing periodontal disease based on enzyme activity.

FIG. 4 is a flowchart showing the flow of a determination method according to an embodiment of the present invention. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 4° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 15° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 22° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 30° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 37.5° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 45° C.) in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement at each temperature of a liquid sample in which bacteria cells have been subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement for each strain of liquid samples in which bacterial cells were subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 4° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 15° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 22° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 30° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 37.5° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 45° C.) in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement at each temperature of a liquid sample in which a bacterial extract was subjected to an enzymatic reaction. FIG. 13 is a diagram showing the results of fluorescence measurement for each strain of liquid samples obtained by enzymatically reacting bacterial cell extracts. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 4° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 15° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 22° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 30° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 37.5° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature 45° C.) in which bacterial cells were subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 4° C.) in which a bacterial cell extract was subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 15° C.) in which a bacterial extract was subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 22° C.) in which a bacterial extract was subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of a liquid sample (liquid temperature: 30° C.) in which a bacterial extract was subjected to an enzymatic reaction at different pH levels. FIG. 13 is a diagram showing the results of fluorescence measurement of liquid samples (liquid temperature 37.5° C.) in which a bacterial extract was subjected to an enzymatic reaction at different temperatures. FIG. 13 is a diagram showing the results of fluorescence measurement of liquid samples (liquid temperature: 45° C.) in which a bacterial extract was subjected to an enzymatic reaction at different temperatures. FIG. 1 is a diagram for explaining the principle of determining the genotype of bacteria causing periodontal disease using bacterial cells. FIG. 1 is a diagram for explaining the principle of discriminating the genotype of bacteria causing periodontal disease using a bacterial cell extract. 1 is a diagram showing a configuration of a fluorescence measuring device according to an embodiment of the present invention. FIG. 2 is a diagram showing a schematic configuration of a control device provided in the fluorescence measuring device. FIG. 4 is a flow chart showing the flow of a discrimination method using a fluorescence measuring device. FIG. 11 is a flow chart showing the flow of a process for determining a genotype using a fluorescence measuring device.

<Method of determination>
First, a determination method according to an embodiment of the present invention will be described with reference to the drawings.

The discrimination method according to this embodiment is a method for discriminating the genotype of bacteria causing periodontal disease. In this discrimination method, it is determined which genotype, among known genetic polymorphisms, the bacteria causing periodontal disease in a collected sample belongs to. The genotype is discriminated based on enzyme activity, the correlation of which with each genotype has been confirmed. The enzyme activity is evaluated by a fluorometric method using a test agent that uses a fluorescently labeled substrate for the enzyme reaction.

The causative bacteria of periodontal disease that are the subject of genotyping include Porphyromonas gingivalis (Pg bacteria). The genotypes to be distinguished include type I (type 1), type II (type 2), type III (type 3), type IV (type 4), and type V (type 5), which are polymorphisms of the fimA gene that codes for pilus proteins. Type Ib, which is a subtype of the fimA gene, is considered to belong to type I.

In the discrimination method according to this embodiment, a liquid sample for fluorescence measurement is prepared by adding bacteria of a periodontal disease causative bacterium of unknown genotype or an extract of the bacteria (specimen) to a liquid test agent containing a fluorescently labeled substrate for an enzyme reaction (fluorescently labeled substrate). The fluorescently labeled substrate is a substrate for a decomposition enzyme produced by the periodontal disease causative bacterium, and dissociates a fluorescent chromophore through an enzyme reaction. The dissociated fluorescent chromophore fluoresces when irradiated with excitation light, and the intensity of the fluorescence emitted from the liquid sample is an index of enzyme activity.

The enzymatic activity of the decomposing enzyme contained in the test sample behaves differently with respect to temperature depending on the genotype of Pg bacteria, so the test sample can be classified into one of the genotypes. The behavior of the enzymatic activity with respect to temperature tends to differ depending on the form of the test sample and the pH of the liquid sample used for fluorescence measurement. Therefore, depending on the form of the test sample, the enzymatic activity is determined by fluorescence measurement under specified temperature conditions and specified pH conditions, and the behavior of the enzymatic activity with respect to temperature is compared.

The specimen used to prepare a liquid sample for fluorescence measurement to distinguish the genotype is the bacteria of the causative bacteria of periodontal disease of unknown genotype, or an extract of the bacteria. For example, dental plaque, gingival exudate, etc. collected from the oral cavity of a subject can be used as is, or as a suspension or the supernatant of a suspension. Generally, in the oral bacterial flora of humans, etc., the causative bacteria of periodontal disease exist in a state where one genotype is dominant. Therefore, the test bacteria collected from a certain subject can be classified into one of the dominant genotypes.

A liquid sample for fluorescence measurement may be prepared by adding bacterial cells contained in dental plaque, gingival exudate, etc. to a liquid test agent containing a fluorescently labeled substrate, or by adding a bacterial cell extract extracted from the bacterial cells. Note that bacterial cell extracts include both extracts obtained by separating the bacterial cells from the residues by cell disruption treatment, etc., and secretions secreted or produced outside the cells by the bacterial cells, so long as they contain a decomposing enzyme that reacts with the fluorescently labeled substrate.

FIG. 1 is a flow chart showing the flow of a determination method according to an embodiment of the present invention.
As shown in FIG. 1, the discrimination method according to this embodiment includes an enzyme reaction step S10, a fluorescence measurement step S20, and a genotype discrimination step S30.

In the enzyme reaction step S10, the bacteria of the causative bacteria of periodontal disease, the genotype of which is unknown, or an extract of the bacteria (specimen) is added to a liquid sample containing a fluorescently labeled substrate and a liquid medium to initiate the enzyme reaction. The fluorescently labeled substrate is a substance in which the substrate of the enzyme reaction by the causative bacteria of periodontal disease is fluorescently labeled with a fluorescent chromophore. The liquid sample to which the specimen has been added to initiate the enzyme reaction becomes the subject of measurement by the fluorometric method.

As a fluorescently labeled substrate, a substance in which the C-terminus of a polypeptide is an L-arginine (Arg) residue and a fluorescent chromophore is bound to the C-terminus of the Arg residue is preferably used. With such a fluorescently labeled substrate, the C-terminus of the Arg residue is specifically digested by gingipain, a type of protease produced by Pg bacteria, making it possible to evaluate the enzymatic activity of Pg bacteria.

The fluorescently labeled substrate may have any amino acid sequence as long as it is recognized by the protease produced by the bacteria that cause periodontal disease. The polypeptide constituting the fluorescently labeled substrate may be composed of any number of amino acids or any type of amino acids. However, from the viewpoint of stabilizing the enzyme reaction and fluorescence measurement, the length of the polypeptide is preferably 3-4 mer.

The fluorescently labeled substrate may be protected at the N-terminus of the polypeptide with a protecting group. Examples of the protecting group include an isobutyloxycarbonyl group (iBoc), a tert-butoxycarbonyl group (Boc), an acetyl group (Ac), a benzoyl group (Bz), and a 9-fluorenylmethoxycarbonyl group (Fmoc).

The fluorescently labeled substrate preferably has aminomethylcoumarin (AMC) bound as a fluorescent chromophore. AMC does not emit fluorescence when in an amide bond, but emits fluorescence only when dissociated, resulting in highly intense fluorescence. Therefore, enzyme activity can be evaluated by highly sensitive fluorescence measurement.

As a fluorescently labeled substrate, the protecting group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (Gly-Gly-Arg-MCA) is preferred, with iBoc-Gly-Gly-Arg-MCA being particularly preferred. Such fluorescently labeled substrates are easily recognized by gingipain produced by Pg bacteria, and high brightness suitable for measurement can be obtained by AMC. This allows for more accurate evaluation of the enzyme activity of Pg bacteria.

The liquid sample to be measured for fluorescence may contain a suitable liquid medium such as a pH buffer solution or water, but preferably contains a pH buffer solution whose main component is either trishydroxymethylaminomethane (Tris), piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), or 2-[4-(2-hydroxyethyl)piperazinyl]ethanesulfonic acid (HEPES). Such a pH buffer solution can suppress the pH fluctuation of the liquid sample to within a range suitable for the gingipain produced by Pg bacteria, allowing the enzyme activity of Pg bacteria to be accurately evaluated.

The liquid sample to be measured for fluorescence preferably contains a pH buffer solution mainly composed of trishydroxymethylaminomethane (Tris). Specific examples of pH buffer solutions mainly composed of Tris include Tris-hydrochloric acid buffer, Tris-acetate buffer, Tris-borate buffer, and Tris-phosphate buffer. A pH buffer solution mainly composed of Tris allows for more accurate evaluation of the enzyme activity of Pg bacteria.

In the fluorescence measurement step S20, excitation light is irradiated onto the liquid sample, and the intensity of fluorescence emitted from the liquid sample is measured. The liquid sample is a solution containing bacteria of the bacteria that cause periodontal disease, or an extract of the bacteria (specimen), and a fluorescently labeled substrate in which the substrate for the enzyme reaction caused by the bacteria that cause periodontal disease is fluorescently labeled. The fluorescently labeled substrate dissociates a fluorescent chromophore in an enzyme reaction caused by a decomposing enzyme produced by the bacteria that cause periodontal disease, and therefore emits fluorescence when irradiated with excitation light in a specified wavelength range. Therefore, by performing fluorescence measurement, it is possible to detect the fluorescence intensity according to the enzyme activity and reaction time of the test bacteria.

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

The wavelength of the fluorescence for measuring the fluorescence intensity is preferably 410 nm or more and 475 nm or less, more preferably 425 nm or more and 465 nm or less, even more preferably 430 nm or more and 455 nm or less, and particularly preferably 435 nm or more and 450 nm or less. With such a wavelength, when the fluorescent chromophore constituting the fluorescently labeled substrate is AMC, the fluorescence can be detected with high sensitivity.

It is preferable to detect fluorescence from a liquid sample after adding the test sample to a liquid test agent containing a fluorescently labeled substrate, and before the fluorescently labeled substrate is completely decomposed and dissociated by an enzyme reaction. It is also preferable to detect fluorescence from a liquid sample before the fluorescence decays due to its fluorescence lifetime. Specifically, it is preferable to detect fluorescence within 10 minutes, and more preferably within 5 minutes, from the start of the enzyme reaction. At such a time, differences in fluorescence intensity according to enzyme activity can be detected with high sensitivity.

In the genotype discrimination process S30, the genotype of the causative bacteria of periodontal disease, whose genotype is unknown, is discriminated based on the intensity of the fluorescence emitted from the liquid sample irradiated with excitation light. The fluorescence intensity value detected by fluorescence measurement indirectly represents the reaction rate of the enzyme reaction, i.e., the proportion of the reacted substrate. Furthermore, the amount of change in the fluorescence intensity over time indirectly represents the reaction rate of the enzyme. Therefore, the genotype can be discriminated based on these fluorescence intensity results.

Here, we will explain the specific principles and methods for identifying the genotype of bacteria that cause periodontal disease, with reference to diagrams.

Fig. 2 shows the fluorescence measurement results of a liquid sample in which bacterial cells were subjected to an enzyme reaction. Fig. 2A shows the result of an enzyme reaction at a liquid temperature of 4°C. Fig. 2B shows the result of an enzyme reaction at a liquid temperature of 15°C. Fig. 2C shows the result of an enzyme reaction at a liquid temperature of 22°C. Fig. 2D shows the result of an enzyme reaction at a liquid temperature of 30°C. Fig. 2E shows the result of an enzyme reaction at a liquid temperature of 37.5°C. Fig. 2F shows the result of an enzyme reaction at a liquid temperature of 45°C.
Figures 2A to 2F show the results of measuring the fluorescence intensity using a fluorescence measuring device after centrifuging a cell suspension of Pg bacteria to collect the precipitate, adding the precipitate containing this bacterial body to a liquid sample and adjusting the pH to 8.0, and then performing an enzymatic reaction under various temperature conditions.

The horizontal axis of Figures 2A to 2F indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a given unit. The thick lines in the figures show the results for Pg bacteria with a fimA gene genotype of type I, the thin lines show the results for Pg bacteria with a genotype of type II, and the dashed lines show the results for Pg bacteria with a genotype of type IV.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per genotype was three.

As shown in Figures 2A to 2F, the results show that the fluorescence intensity differs for each genotype of the fimA gene. The slope of the curve corresponding to the time change in fluorescence intensity tends to be greater for type IV than for type I, and greater for type II than for type IV. When the liquid sample was adjusted to pH 8.0 and the liquid temperature to 4 to 37.5°C, the difference in the time change in fluorescence intensity for types II and IV was greater compared to when the liquid temperature was adjusted to 45°C. It can be seen that enzyme activity differs for each genotype of the bacteria that causes periodontal disease, and that the behavior of enzyme activity with respect to temperature also differs.

According to the results shown in Figures 2A to 2F, when the bacterial cells themselves are added to a liquid sample containing a fluorescently labeled substrate, the fluorescence intensity of types II and IV increases when the enzyme reaction is carried out in a liquid sample adjusted to a pH of 8.0 and a liquid temperature of 4 to 37.5°C, improving the accuracy of genotype discrimination. It can also be seen that types II and IV have high enzyme activity at liquid temperatures of 4 to 37.5°C and are easily inactivated at a liquid temperature of 45°C, whereas type I has low enzyme activity over the entire range of liquid temperatures from 4 to 45°C.

FIG. 3A is a diagram showing the results of fluorescence measurement at each temperature of a liquid sample in which bacteria were subjected to an enzymatic reaction.
FIG. 3A shows the results of measuring the fluorescence intensity using a fluorescence measuring device after centrifuging a cell suspension of Pg bacteria, whose fimA gene genotype is type II, collecting the precipitate, adding this precipitate containing the bacteria, and adjusting the pH of the liquid sample to 8.0 and carrying out an enzymatic reaction under various temperature conditions.

The horizontal axis in the figure indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a specified unit. The thick dashed line in the figure indicates the result at a liquid temperature of 4°C, the dashed dotted line indicates the result at a liquid temperature of 15°C, the thin dashed line indicates the result at a liquid temperature of 22°C, the dotted line indicates the result at a liquid temperature of 30°C, the thick solid line indicates the result at a liquid temperature of 37.5°C, and the thin solid line indicates the result at a liquid temperature of 45°C.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per temperature was three.

As shown in Figure 3A, the fluorescence intensity varied depending on the temperature of the liquid sample. The fluorescence intensity value per measurement time and the amount of change in initial fluorescence intensity over time tended to be higher at liquid temperatures between 4 and 37.5°C compared to a liquid temperature of 45°C. A liquid temperature of 37.5°C showed a particularly large amount of change over time, indicating that maximum enzyme activity was obtained at a liquid temperature of around 37.5°C.

According to the results shown in Figure 3A, when the bacterial cells themselves are added to a liquid sample containing a fluorescently labeled substrate, a relatively high fluorescence intensity is obtained when the enzyme reaction is carried out in a liquid sample adjusted to a pH of around 8.0 and a liquid temperature of 4 to 37.5°C, and this improves the accuracy of genotype discrimination based on comparison of fluorescence intensity.

FIG. 3B shows the results of fluorescence measurement for each strain of liquid samples in which the bacterial cells were subjected to an enzymatic reaction.
Figure 3B shows the results of measuring the fluorescence intensity using a fluorometer after centrifuging cell suspensions of different strains of Pg bacteria to collect the precipitates, adding the precipitates containing the bacterial cells to a liquid sample and adjusting the pH to 8.0 and carrying out an enzymatic reaction at 37.5°C.

The horizontal axis in the figure indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a given unit. The long dashed line in the figure indicates the result of the 33277 strain, whose genotype of the fimA gene is type I, the dashed line indicates the result of the TDC60 strain, whose genotype is type II, the short dashed line indicates the result of the W83 strain, whose genotype is type IV, the thick solid line indicates the result of the 275 strain, whose genotype is type II, and the thin solid line indicates the result of the 268 strain, whose genotype is type II. The 275 and 268 strains were isolated from subgingival dental plaque (see Hiroyuki Asano et al., Journal of Periodontology, 2003, Vol. 74, 9, p.1355-1360).

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per strain was one.

As shown in Figure 3B, the results show that the fluorescence intensity varies depending on the genotype of the fimA gene. The TDC60, 275, and 268 strains, which have a fimA gene genotype of type II, show significantly greater fluorescence intensity values per measurement time and temporal change in fluorescence intensity than the 33277 strain, which has a type I genotype, and the W83 strain, which has a type IV genotype.

The results shown in Figure 3B show that the enzymatic activity of the decomposition enzyme produced by Pg bacteria differs depending on the genotype of the bacteria that causes periodontal disease, and when the bacterial cells themselves are used as a test sample, the fluorescence intensity values and the amount of change in fluorescence intensity over time differ greatly between type II and types I and IV. It can be said that it is possible to distinguish genotypes, including type II, based on the results of fluorescence measurements using the bacterial cells themselves of any strain.

Fig. 4 shows the fluorescence measurement results of a liquid sample in which a bacterial extract was subjected to an enzyme reaction. Fig. 4A shows the result of an enzyme reaction at a liquid temperature of 4°C. Fig. 4B shows the result of an enzyme reaction at a liquid temperature of 15°C. Fig. 4C shows the result of an enzyme reaction at a liquid temperature of 22°C. Fig. 4D shows the result of an enzyme reaction at a liquid temperature of 30°C. Fig. 4E shows the result of an enzyme reaction at a liquid temperature of 37.5°C. Fig. 4F shows the result of an enzyme reaction at a liquid temperature of 45°C.
Figures 4A to 4F show the results of measuring the fluorescence intensity using a fluorescence measuring device after centrifuging a Pg cell suspension to collect the supernatant, adding this supernatant, which is an extract of the bacterial cell, to a liquid sample adjusted to pH 8.0 and carrying out an enzymatic reaction under various temperature conditions.

The horizontal axis of Figures 4A to 4F indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a given unit. The thick lines in the figures show the results for Pg bacteria with a fimA gene genotype of type I, the thin lines show the results for Pg bacteria with a genotype of type II, and the dashed lines show the results for Pg bacteria with a genotype of type IV.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per genotype was three.

As shown in Figures 4A to 4F, the results show that the fluorescence intensity differs for each genotype of the fimA gene. The slope of the curve corresponding to the change in fluorescence intensity over time shows a tendency for type IV to be greater than type II, and type I to be greater than type IV, in contrast to the case of bacterial bodies. When the liquid sample was adjusted to pH 8.0 and the liquid temperature to 15 to 37.5°C, the difference in the change in fluorescence intensity over time for types I and IV was greater compared to when the liquid temperature was adjusted to 4°C or 45°C. It can be seen that enzyme activity differs for each genotype of the bacteria that causes periodontal disease, and that the behavior of enzyme activity with respect to temperature also differs.

The results shown in Figures 4A to 4F show that when a bacterial extract is added to a liquid sample containing a fluorescently labeled substrate, the fluorescence intensity of type I increases and the accuracy of genotype discrimination improves when the enzyme reaction is carried out in a liquid sample adjusted to a liquid temperature of 15 to 37.5°C at a pH of around 8.0. It is also clear that types I and IV have high enzyme activity at liquid temperatures of 15 to 37.5°C and are easily inactivated at liquid temperatures of 4°C and 45°C, whereas type II has low enzyme activity over the entire range of liquid temperatures from 4 to 45°C.

FIG. 5A shows the results of fluorescence measurement at each temperature of a liquid sample in which a bacterial extract was subjected to an enzymatic reaction.
FIG. 5A shows the results of measuring the fluorescence intensity using a fluorometer after centrifuging a cell suspension of Pg bacteria, whose fimA gene genotype is type I, collecting the supernatant, and adding this supernatant, which is an extract of the bacterial cell, to a liquid sample adjusted to pH 8.0 and allowing an enzyme reaction to occur under various temperature conditions.

The horizontal axis in the figure indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a specified unit. The thick dashed line in the figure indicates the result at a liquid temperature of 4°C, the dashed dotted line indicates the result at a liquid temperature of 15°C, the thin dashed line indicates the result at a liquid temperature of 22°C, the dotted line indicates the result at a liquid temperature of 30°C, the thick solid line indicates the result at a liquid temperature of 37.5°C, and the thin solid line indicates the result at a liquid temperature of 45°C.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per temperature was three.

As shown in Figure 5A, the fluorescence intensity varied depending on the temperature of the liquid sample. The fluorescence intensity value per measurement time and the amount of change in fluorescence intensity over time tended to be higher at liquid temperatures of 15 to 37.5°C compared to liquid temperatures of 4°C and 45°C. Liquid temperatures of 22°C, 30°C, and 37.5°C showed particularly large amounts of change over time, indicating that maximum enzyme activity was obtained at liquid temperatures of around 22 to 37.5°C.

According to the results shown in Figure 5A, when the enzyme reaction is carried out in a liquid sample adjusted to a pH of around 8.0 and a liquid temperature of 15 to 37.5°C, a relatively high fluorescence intensity is obtained in the method of adding a bacterial extract to a liquid sample containing a fluorescently labeled substrate, and it can be said that the accuracy of genotype discrimination based on comparison of fluorescence intensity is improved.

FIG. 5B shows the results of fluorescence measurement for each strain of liquid samples obtained by enzymatically reacting bacterial extracts.
FIG. 5B shows the results of measuring the fluorescence intensity using a fluorometer after adding the supernatant, which is a bacterial cell extract, to a liquid sample that was adjusted to pH 8.0 and subjected to an enzymatic reaction at 37.5°C after centrifuging cell suspensions of different Pg strains.

The horizontal axis in the figure indicates the measurement time [min] calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity in a specified unit. The long dashed line in the figure indicates the result for strain 33277, whose fimA gene genotype is type I, the dashed line indicates the result for strain TDC60, whose genotype is type II, the short dashed line indicates the result for strain W83, whose genotype is type IV, the thick solid line indicates the result for strain 275, whose genotype is type II, and the thin solid line indicates the result for strain 268, whose genotype is type II.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per strain was one.

As shown in Figure 5B, the results show that the fluorescence intensity varies depending on the genotype of the fimA gene. The 33277 strain, whose fimA gene genotype is type I, and the W83 strain, whose fimA gene genotype is type IV, show significantly larger fluorescence intensity values per measurement time and changes in fluorescence intensity over time than the TDC60, 275, and 268 strains, whose fimA gene genotypes are type II. Furthermore, the 33277 strain, which is type I, shows larger fluorescence intensity values and changes in fluorescence intensity over time than the W83 strain, which is type IV.

The results shown in Figure 5B show that the enzymatic activity of the degradative enzyme produced by Pg bacteria differs depending on the genotype of the bacteria that causes periodontal disease, and when bacterial cell extracts are used as samples, it can be seen that the fluorescence intensity values and the amount of change in fluorescence intensity over time differ significantly between types I, II, and IV. It can be said that it is possible to distinguish genotypes, including type I, based on the results of fluorescence measurement using bacterial cell extracts of any strain.

Fig. 6 shows the fluorescence measurement results of a liquid sample in which bacterial cells were subjected to an enzymatic reaction with different pH values. Fig. 6A shows the results of an enzymatic reaction at 4°C. Fig. 6B shows the results of an enzymatic reaction at 15°C. Fig. 6C shows the results of an enzymatic reaction at 22°C. Fig. 6D shows the results of an enzymatic reaction at 30°C. Fig. 6E shows the results of an enzymatic reaction at 37.5°C with different pH values. Fig. 6F shows the results of an enzymatic reaction at 45°C.
Figures 6A to 6F show the results of measuring the fluorescence intensity using a fluorescence measuring device after centrifuging cell suspensions of Pg bacteria of each genotype, collecting the precipitate, adding the precipitate containing the bacteria to liquid samples, and adjusting the pH to various conditions and carrying out an enzymatic reaction under various temperature conditions.

The horizontal axis in Figures 6A to 6F indicates the temperature of the liquid sample [°C], and the vertical axis indicates the amount of change in fluorescence intensity per minute. The amount of change in fluorescence intensity over time was measured after the start of the enzyme reaction and converted into the amount of change per minute. The plots marked with ● in the figures show the results for the 33277 strain, whose fimA gene genotype is type I, the plots marked with ■ show the results for the TDC60 strain, whose genotype is type II, and the plots marked with ▲ show the results for the W83 strain, whose genotype is type IV.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per strain was three.

As shown in Figure 6A, when the liquid temperature is 4°C, the time change values for each genotype are similar at pH 7.0 and pH 7.5. On the other hand, at pH 7.8 to 8.5, the time change values for types II and IV are significantly greater than type I. In particular, at pH 7.8 to 8.0, the time change value for type II is significantly greater than not only type I but also type IV.

As shown in Figure 6B, when the liquid temperature is 15°C, at pH 7.0 to 8.5, the time change values of types II and IV are significantly greater than type I. At pH 7.0 to 7.8, the time change value of type II is slightly greater than type IV, but there is no significant difference in the time change values. On the other hand, at pH 8.0 to 8.5, the time change value of type II is significantly greater than not only type I but also type IV.

As shown in Figure 6C, when the liquid temperature is 22°C, at pH 7.5, the time change values for each genotype are similar. On the other hand, at pH 7.0 and pH 7.8 to 8.5, the time change values for types II and IV are significantly greater than type I. In particular, at pH 8.0 to 8.5, the time change value for type II is significantly greater than not only type I but also type IV.

As shown in Figure 6D, when the liquid temperature is 30°C, the time change value of type I gradually increases from pH 7.0 to 8.0, but drops significantly at pH 8.5. At pH 7.0, the time change values for each genotype are similar. On the other hand, at pH 7.5 to 8.5, the time change values of types II and IV are significantly greater than type I. Furthermore, at pH 7.5 to 8.5, the time change value of type II is significantly greater than not only type I but also type IV.

As shown in Figure 6E, when the liquid temperature is 37.5°C, the time change values of types I and IV gradually increase from pH 7.0 to 8.0, but drop significantly at pH 8.5. At pH 7.5, the time change values for each genotype are similar to each other. On the other hand, at pH 7.0 and pH 7.8 to 8.5, the time change values of types II and IV are significantly greater than type I. In particular, at pH 7.8 to 8.5, the time change value of type II is significantly greater than not only type I but also type IV.

As shown in Figure 6F, when the liquid temperature is 45°C, the time change values for each genotype are similar at pH 7.5 and pH 8.5. On the other hand, at pH 7.0 and pH 7.8-8.0, the time change values for types II and IV are significantly greater than type I. In particular, at pH 7.8, the time change value for type II is significantly greater than not only type I but also type IV.

According to the results shown in Figures 6A to 6F, when enzyme-encapsulating bacteria are subjected to an enzymatic reaction in a liquid sample adjusted to a pH of around 7.0 or higher and a pH of 8.5 or lower, and adjusted to 4 to 45°C, the fluorescence intensity of type II tends to be higher than that of types I and IV, and the fluorescence intensity of type IV tends to be higher than that of type I. Therefore, it can be said that it is possible to distinguish between types II and I or IV, and between type IV and types I or II, based on the fluorescence measurement results. It can also be seen that the behavior of enzyme activity for each genotype differs greatly between temperature ranges lower than 38 to 45°C and temperature ranges higher than 38 to 45°C.

Fig. 7 shows the fluorescence measurement results of liquid samples in which bacterial cell extracts were subjected to an enzymatic reaction at different pH levels. Fig. 7A shows the results of the enzymatic reaction at 4°C. Fig. 7B shows the results of the enzymatic reaction at 15°C. Fig. 7C shows the results of the enzymatic reaction at 22°C. Fig. 7D shows the results of the enzymatic reaction at 30°C. Fig. 7E shows the results of the enzymatic reaction at 37.5°C. Fig. 7F shows the results of the enzymatic reaction at 45°C.
Figures 7A to 7F show the results of measuring the fluorescence intensity using a fluorescence measuring device after centrifuging a cell suspension of Pg bacteria of each genotype, collecting the supernatant, and adding this supernatant, which is an extract of the bacterial cell, to liquid samples adjusted to various pH conditions and carrying out an enzymatic reaction under various temperature conditions.

The horizontal axis in Figures 7A to 7F indicates the temperature of the liquid sample [°C], and the vertical axis indicates the amount of change in fluorescence intensity per minute. The amount of change in fluorescence intensity over time was measured after the start of the enzyme reaction and converted into the amount of change per minute. The plots marked with ● in the figures show the results for the 33277 strain, whose fimA gene genotype is type I, the plots marked with ■ show the results for the TDC60 strain, whose genotype is type II, and the plots marked with ▲ show the results for the W83 strain, whose genotype is type IV.

The fluorescently labeled substrate used was isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The number of liquid samples per strain was three.

As shown in Figure 7A, when the liquid temperature is 4°C, the time change values of types I, IV, and II gradually increase from pH 7.0 to 8.0, but decrease at pH 8.5. At pH 8.0, the time change values for each genotype are approximately the same. On the other hand, at pH 7.0 to 7.8 and pH 8.5, the time change values of types I and IV are significantly greater than type II. Furthermore, at pH 7.0, the time change value of type I is significantly greater than not only type II but also type IV.

As shown in Figure 7B, when the liquid temperature is 15°C, the time change values of types I, IV, and II gradually increase from pH 7.0 to 8.0, but drop significantly at pH 8.5. The time change value of type I is significantly larger at pH 8.0. At pH 7.0 to 7.8, the time change values of each genotype are approximately the same. On the other hand, at pH 8.0 to 8.5, the time change value of type I is significantly larger than types IV and II.

As shown in Figure 7C, when the liquid temperature is 22°C, the time change values of types I, IV, and II gradually increase from pH 7.0 to 8.0, but drop significantly at pH 8.5. The time change value of type I is significantly larger at pH 8.0. At pH 7.0 to 7.5, the time change values of each genotype are approximately the same. On the other hand, at pH 7.8 to 8.5, the time change value of type I is significantly larger than types IV and II.

As shown in Figure 7D, when the liquid temperature is 30°C, the time change values of types I, IV, and II gradually increase from pH 7.0 to 8.0, but drop significantly at pH 8.5. The time change value of type I is significantly larger at pH 7.8 to 8.0. At pH 7.0 to 8.5, the time change values of types I and IV are significantly larger than type II. Furthermore, at pH 7.5 and pH 8.0, the time change value of type I is significantly larger than not only type II but also type IV.

As shown in Figure 7E, when the liquid temperature is 37.5°C, the time change values of types I, IV, and II gradually increase from pH 7.0 to 8.0, but drop significantly at pH 8.5. The time change value of type I is significantly larger at pH 8.0. At pH 7.0, the time change values of each genotype are approximately the same. On the other hand, at pH 7.5 to 8.5, the time change value of type I is significantly larger than types IV and II. Furthermore, at pH 7.8 to 8.0, the time change value of type I is significantly larger than not only type II but also type IV.

As shown in Figure 7F, when the liquid temperature is 45°C, the time change values of types IV and II gradually increase from pH 7.0 to 8.0, but decrease at pH 8.5. At pH 8.0, the time change values for each genotype are approximately the same. On the other hand, at pH 7.0 to 7.8 and pH 8.5, the time change value of type I is significantly greater than that of types IV and II.

According to the results shown in Figures 7A to 7F, when the enzyme-producing bacterial extract is subjected to an enzymatic reaction in a liquid sample adjusted to pH 7.0 or higher but lower than pH 8.5 and adjusted to a temperature between 15°C and 45°C, the fluorescence intensity of type I tends to be higher than that of types IV and II, and the fluorescence intensity of type IV tends to be higher than that of type II. Therefore, it can be said that it is possible to distinguish between types I and IV or II, and between type IV and types I and II, based on the fluorescence measurement results. It can also be seen that the behavior of the enzyme activity for each genotype differs greatly between temperature ranges lower than 4 to 15°C and higher than 38 to 45°C, and temperature ranges higher than 4 to 15°C and lower than 38 to 45°C.

FIG. 8 is a diagram for explaining the principle of determining the genotype of bacteria causing periodontal disease using bacterial cells.
8, the horizontal axis indicates the time calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity. The dashed line in the figure indicates a specific example of the fluorescence measurement result of a liquid sample containing bacteria (specimen) of a periodontal disease causative bacterium of unknown genotype.

The thick solid line in the figure shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype I, the thin solid line shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype II, and the dashed line shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype IV. Such fluorescence measurement results are obtained when the temperature of the liquid sample is in the temperature range below 38-45°C. In the temperature range above 38-45°C, the fluorescence measurement results of types II and IV form curves that approximate the fluorescence measurement results of type I.

As shown in Figure 8, when a liquid sample containing bacteria of a known genotype that causes periodontal disease is subjected to fluorescence measurement, if the enzyme activity of the bacteria that causes periodontal disease and their extracellular production and secretion capabilities vary depending on the genotype, different fluorescence intensities are detected for each genotype depending on the temperature and pH of the liquid sample. When performing fluorescence measurement, variations occur due to the amount of bacteria, amount of enzyme, reaction time, measurement error, etc., so when multiple fluorescence measurements are performed, fluorescence measurement results that are distributed over a certain range are obtained, as shown by the shaded area in the figure. When fluorescence measurements are performed on multiple liquid samples of known genotypes and the results are subjected to regression analysis, etc., a representative value for the measurement value group for each genotype can be obtained, as shown by the curves (type I line, type II line, type IV line) in the figure.

On the other hand, when a liquid sample containing bacteria of a periodontal disease causative bacterium of unknown genotype is subjected to fluorescence measurement, the measurement result obtained is close to the representative curve of one of the genotypes (type I line, type II line, type IV line), as shown by the dashed dotted line in the figure. Figure 8 shows a measurement result close to the type II line. Such a measurement result is obtained because the specimen collected from the subject is in a state in which one of the genotypes is dominant, and the enzyme activity and the degree of extracellular production and secretion ability of the enzyme are similar to those of one of the genotypes.

Therefore, when a predetermined reaction time has elapsed after the start of the enzyme reaction (e.g., time _T1 ), the fluorescence intensity of the liquid sample is measured, and the fluorescence intensity value (e.g., fluorescence intensity _I0 ) of a liquid sample containing bacteria of the causative bacteria of periodontal disease whose genotype is unknown is compared with the fluorescence intensity value (e.g., fluorescence intensity _I2 , _I4 , or the boundary line of the shaded area in the figure) of a liquid sample containing bacteria of the causative bacteria of periodontal disease whose genotype is known, per the same reaction time, so that the genotype of the test sample can be determined.

In this type of discrimination method, when comparing a test area containing a test material with an unknown genotype with a control area containing bacteria that cause periodontal disease with a known genotype, it is necessary to match the reaction conditions for the enzyme reaction and the measurement system for fluorescence measurement, but the genotype can be discriminated by simply comparing the fluorescence intensity values.

Alternatively, the fluorescence intensity of the liquid sample can be measured over time at predetermined time intervals (e.g., in a small section around time _T1 ) after the start of the enzyme reaction, and the time change (slope), which is the time derivative of the fluorescence intensity, can be calculated. By comparing the time change in fluorescence intensity of a liquid sample containing bacteria that cause periodontal disease and whose genotype is unknown (e.g., the slope of the tangent at the intersection of _T1 - _I0 ) with the time change in fluorescence intensity of a liquid sample containing bacteria that cause periodontal disease and whose genotype is known (e.g., the slope of the tangent at the intersection of _T1 - _I2 , the slope of the tangent at the intersection of _T1 - _I4 , or the slope of the tangent to the boundary line of the shaded area in the figure), the genotype of the test sample can be determined.

In this type of discrimination method, calculation of the amount of change over time (slope) is required, but the reaction time of the enzyme reaction does not necessarily need to be the same, and the results of the fluorescence measurement are less likely to have systematic measurement errors, making it possible to make accurate comparisons. The amount of change over time in fluorescence intensity can be compared for the same reaction time, or the maximum values during the enzyme reaction can be compared.

FIG. 9 is a diagram for explaining the principle of determining the genotype of bacteria causing periodontal disease using bacterial cell extracts.
9, the horizontal axis indicates the time calculated from the start of the enzyme reaction, and the vertical axis indicates the fluorescence intensity. The dashed line in the figure indicates a specific example of the fluorescence measurement result of a liquid sample containing a bacterial extract (specimen) extracted from a causative bacterium of periodontal disease whose genotype is unknown.

In addition, the thick solid line in the figure shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype II, the thin solid line shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype I, and the dashed line shows a specific example of the fluorescence measurement results obtained with Pg bacteria of genotype IV. Such fluorescence measurement results are obtained when the temperature of the liquid sample is in the temperature range higher than 4 to 15°C and lower than 38 to 45°C. In the temperature range lower than 4 to 15°C and higher than 38 to 45°C, the fluorescence measurement results of types I and IV form curves that approximate the fluorescence measurement results of type II.

As shown in Figure 9, when fluorescence measurements are performed on a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease with a known genotype, if the enzyme activity of the causative bacterium of periodontal disease differs depending on the genotype, different fluorescence intensities are detected for each genotype depending on the temperature and pH of the liquid sample, just as when bacterial cells are used (see Figure 8). However, when bacterial cell extracts are used, the influence of the enzyme's extracellular production and secretion capabilities is reduced, so the measured values of fluorescence intensity for each genotype tend to differ from those when bacterial cells are used.

On the other hand, when fluorescence measurements are performed on a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease whose genotype is unknown, the measurement results obtained are close to the representative curves of one of the genotypes (type I line, type II line, type IV line), as shown by the dashed dotted lines in the figure. When a bacterial cell extract is used, the fluorescence intensity of type I and type II have an inverse relationship compared to when bacteria are used, so the smaller the measured fluorescence intensity value, the closer it is to the type II curve, and the greater the measured fluorescence intensity value, the closer it is to the type I curve.

Therefore, when a bacterial cell extract is used, as in the case of using bacterial cells (see Figure 8), the fluorescence intensity of the liquid sample is measured when a predetermined reaction time has elapsed after the start of the enzyme reaction (e.g., time _T1 ), and the fluorescence intensity value (e.g., fluorescence intensity _I0 ) of a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease whose genotype is unknown is compared with the fluorescence intensity value (e.g., fluorescence intensity _I2 , _I4 , or the boundary line of the shaded area in the figure) of a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease whose genotype is known, per the same reaction time, thereby making it possible to determine the genotype of the test sample.

In addition, the fluorescence intensity of the liquid sample is measured over time at predetermined time intervals (e.g., in a small section near time _T1 ) after the start of the enzyme reaction, and the time change (slope), which is the time derivative of the fluorescence intensity, is calculated. By comparing the time change in the fluorescence intensity of a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease of unknown genotype (e.g., the slope of the tangent at the intersection of _T1 - _I0 ) with the time change in the fluorescence intensity of a liquid sample containing a bacterial cell extract extracted from a causative bacterium of periodontal disease of known genotype (e.g., the slope of the tangent at the intersection of _T1 - _I2 , the slope of the tangent at the intersection of _T1 - _I4 , or the slope of the tangent at the boundary line of the shaded area in the figure), the genotype of the test sample can be determined.

For example, the genotype determination in the genotype determination step S30 can be performed by preparing a liquid sample in a test area consisting of a liquid sample containing a causative bacterium of periodontal disease of unknown genotype or a bacterial cell extract (test material), and a liquid sample in a control area consisting of a causative bacterium of periodontal disease of known genotype or a liquid sample containing a bacterial cell extract, adjusting the liquid temperatures to the same for both and allowing the enzyme reaction to occur, and then comparing the fluorescence measurement results of the liquid sample in the test area and the fluorescence measurement results of the liquid sample in the control area. The liquid sample in the test area and the liquid sample in the control area are adjusted to substantially the same conditions for the pH, the amount of causative bacterium of periodontal disease or the test material, the concentration of the fluorescently labeled substrate, etc., and allowed to react with the enzyme.

The control area can be composed of any number of liquid samples, one or more, but is preferably composed of a large number of liquid samples. The control area may contain periodontal disease causative bacteria of known genotypes or liquid samples containing their bacterial cell extracts, as well as periodontal disease causative bacteria of unknown genotypes or liquid samples containing their bacterial cell extracts. However, from the viewpoint of reliably distinguishing the genotype, it is preferable that the control area is composed only of periodontal disease causative bacteria of known genotypes or liquid samples containing their bacterial cell extracts.

The control area may contain bacteria that cause periodontal disease and have a known genotype, or a bacterial cell extract thereof, of one genotype or a bacterial cell extract thereof, or may contain bacteria of multiple genotypes or a bacterial cell extract thereof. For example, if the control area contains only type I, it is possible to determine whether the test bacteria is type I or not. If the control area contains types I to V, it is possible to determine which genotype the test substance is.

Controls can be prepared using bacteria causing periodontal disease with known genotypes, or their cell extracts, such as bacteria from strains whose genotypes have been determined in advance by fluorescence measurement, or their cell extracts, or bacteria from deposited or isolated strains that are generally available through distribution, etc.

Specific examples of deposited and isolated strains include ATCC_33277 and ATCC_BAA-1703 (FDC381) strains, which have type I fimA genes; JCM_19600 (TDC60), 275 (HG184), and 268 strains, which have type II fimA genes; ATCC_49417 (RB22D-1) strains, which have type III fimA genes; ATCC_BAA-308 (W83) and ATCC_53978 (W50) strains, which have type IV fimA genes; and HNA99 strain, which has type V fimA genes.

The liquid sample to be measured for fluorescence is preferably adjusted to a neutral pH or higher and a pH of 8.5 or lower before the fluorescence measurement and then subjected to an enzymatic reaction. In this specification, neutral means a pH of 6.8 or higher and a pH of 7.2 or lower. The pH of the liquid sample is preferably 8.4 or lower, more preferably 8.3 or lower, even more preferably 8.2 or lower, and even more preferably 8.1 or lower. When a bacterial cell extract is used, the pH of the liquid sample is preferably 7.5 or higher, more preferably 7.8 or higher. By adjusting the pH to such a level, the enzyme activity of gingipain produced by Pg bacteria can be appropriately obtained, so that the genotype of the fimA gene of Pg bacteria can be more accurately determined. On the other hand, when bacterial cells are used, the pH of the liquid sample is preferably neutral. When bacterial cells are used, the liquid sample can be easily prepared without adjusting the pH.

The liquid sample to be measured for fluorescence is preferably adjusted to the same predetermined temperature for each of the test and control sections, and the fluorescence is preferably measured under constant temperature control adjusted to the predetermined temperature. The temperature of the liquid sample is preferably 4°C or higher and 45°C or lower. When a bacterial cell extract is used, the temperature of the liquid sample is preferably 25°C or higher, more preferably 30°C or higher, even more preferably 34°C or higher, and even more preferably 36°C or higher. Also, it is preferably 40°C or lower, more preferably 39°C or lower, and even more preferably 38°C or lower. On the other hand, when a bacterial cell is used, the temperature of the liquid sample is preferably 4°C or higher, more preferably 10°C or higher, even more preferably 15°C or higher, even more preferably 18°C or higher, and even more preferably 21°C or higher. Also, it is preferably 37°C or lower, more preferably 30°C or lower. When the pH is neutral, it is preferably 22°C or higher and 37.5 or lower, and particularly preferably 30°C. When the temperature is controlled in this manner, the genotype of the fimA gene of Pg bacteria can be accurately determined.

Specifically, the comparison of the fluorescence measurement results of the test area with the fluorescence measurement results of the control area can be performed by comparing the fluorescence intensity value or the amount of change in fluorescence intensity over time of a liquid sample containing the causative bacteria of periodontal disease or a bacterial cell extract (specimen) of unknown genotype measured in the test area with the fluorescence intensity value or the amount of change in fluorescence intensity over time of a liquid sample containing the causative bacteria of periodontal disease or a bacterial cell extract of known genotype measured in the control area. When making the comparison, the fluorescence intensity values or the amount of change in fluorescence intensity over time are compared among the fluorescence measurement results obtained by performing an enzyme reaction at the same liquid temperature.

When the results of the fluorescence measurement in the test area are compared with those of the control area, and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured in the test area is identical to or similar to the fluorescence intensity value or the amount of change in fluorescence intensity over time measured in the control area, it can be determined that the fimA gene of the periodontal disease causative bacterium in the liquid sample in the test area is of the same genotype as the fimA gene of the periodontal disease causative bacterium of which the genotype is known in the liquid sample in the control area.

On the other hand, when the result of comparing the fluorescence measurement results of the test area with the fluorescence measurement results of the control area shows that the fluorescence intensity value or the amount of change in fluorescence intensity over time measured in the test area is not identical and not close to the fluorescence intensity value or the amount of change in fluorescence intensity over time measured in the control area, it can be determined that the fimA gene of the periodontal disease causative bacterium in the liquid sample in the test area is of a different genotype from the fimA gene of the periodontal disease causative bacterium of which the genotype is known in the liquid sample in the control area.

The fluorescence measurement results of the test area can be compared with those of the control area by, for example, comparing the measurements themselves or comparing representative values such as average values. For example, the measurement values measured in the test area can be compared with representative values such as average values measured in the control area, and when the difference between the measurement values in the test area and the representative value in the control area is within a range of ±30% of the representative value in the control area, it can be determined that the fluorescence measurement results are similar.

Alternatively, the genotype determination in the genotype determination step S30 can be performed by preparing a sample group consisting of multiple liquid samples including the liquid sample to be determined that contains the causative bacteria of periodontal disease or a bacterial cell extract (specimen) of unknown genotype, adjusting the liquid temperature to the same for each sample and performing an enzymatic reaction, and then comparing the fluorescence measurement results of the liquid samples in the sample group. Note that the liquid sample to be determined and the other liquid samples are subjected to the enzymatic reaction under substantially the same conditions, such as pH, amount of causative bacteria of periodontal disease or bacterial cell extract, and concentration of fluorescently labeled substrate.

The sample group can be composed of any number of liquid samples, two or more, but is preferably composed of a large number of liquid samples. As long as the sample group includes a liquid sample to be identified that contains a causative bacterium or bacterial cell extract (specimen) of periodontal disease of unknown genotype, the sample group may be composed only of liquid samples containing causative bacterium or bacterial cell extract of periodontal disease of unknown genotype, or may be composed of a combination of liquid samples containing causative bacterium or bacterial cell extract of periodontal disease of unknown genotype and liquid samples containing causative bacterium or bacterial cell extract of periodontal disease of known genotype.

The sample group preferably contains strains or cell extracts of multiple genotypes as bacteria or cell extracts that cause periodontal disease and have known genotypes. For example, if the control group contains types I to V, the fluorescence measurement results will produce five groups of measurement values in which the trends in the time change in fluorescence intensity differ for each genotype. Therefore, by determining the similarity between the fluorescence measurement results of the test sample and these groups of measurement values, it is possible to determine which genotype the test sample belongs to.

As in the case of comparing a test group with a control group, the liquid sample to be measured for fluorescence is preferably adjusted to a neutral pH or higher and a pH of 8.5 or lower before fluorescence measurement, and then subjected to an enzymatic reaction. In addition, the liquid samples to be measured for fluorescence are preferably adjusted to the same predetermined temperature for each liquid sample in the test group, and the fluorescence is measured under constant temperature control adjusted to the predetermined temperature. The temperature of the liquid sample depends on the type of test specimen, but is preferably 4°C or higher and 45°C or lower.

Specifically, the comparison of the fluorescence measurement results in a sample group can be performed by comparing the fluorescence intensity value or the amount of change in fluorescence intensity over time of the liquid sample to be distinguished, which contains the causative bacteria of periodontal disease or a bacterial extract (specimen) of unknown genotype in the sample group, with a group of measurements of the fluorescence intensity values or the amount of change in fluorescence intensity over time obtained for multiple liquid samples in the sample group. When making the comparison, the fluorescence intensity values or the amount of change in fluorescence intensity over time are compared among the fluorescence measurement results obtained by performing an enzyme reaction at the same liquid temperature.

When bacterial cells are used to prepare a liquid sample, if the fluorescence measurement results (measurement value group) in the sample group are compared and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be discriminated whose genotype is unknown in the sample group is classified into the top measurement value group of the measurement values of the fluorescence intensity values or the amount of change in fluorescence intensity over time measured for multiple liquid samples in the sample group, it can be determined that the fimA gene of the periodontal disease-causing bacteria in the liquid sample is type II. On the other hand, when a bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the periodontal disease-causing bacteria in the liquid sample classified into the top measurement value group is type I.

As shown by dotted shading in Figure 8 and spotted shading in Figure 9, the top measurement value group is the measurement value group into which the maximum value of fluorescence intensity is classified among all genotypes. Similarly, the top measurement value group is also the measurement value group into which the maximum value of the temporal change (slope) of fluorescence intensity is classified. Therefore, if bacterial cells are used to prepare the liquid sample and the sample group contains types II, I, and IV, the top measurement value group will be type II. On the other hand, if bacterial cell extracts are used to prepare the liquid sample and the sample group contains types I, IV, and II, the top measurement value group will be type I.

In addition, when bacterial cells are used to prepare a liquid sample, or when a bacterial cell extract is used to prepare a liquid sample, if the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be discriminated, whose genotype is unknown in the sample group, is compared with the fluorescence measurement results (measurement value group) in the sample group and is classified into the second highest measurement value group among the measurement values of the fluorescence intensity values or the amount of change in fluorescence intensity over time measured for multiple liquid samples in the sample group, it can be determined that the fimA gene of the bacteria causing periodontal disease in the liquid sample is type IV.

As shown by the diagonal shading in Figures 8 and 9, the second highest measurement value group is the next highest measurement value group among all genotypes after the measurement value group classified by the maximum value of fluorescence intensity. Similarly, the second highest measurement value group is also the next highest measurement value group after the measurement value group classified by the maximum value in terms of the temporal change (slope) of fluorescence intensity. Therefore, if bacterial cells are used to prepare the liquid sample and the sample group contains types II, IV, and I, the second highest measurement value group will be type IV. Also, if bacterial cell extracts are used to prepare the liquid sample and the sample group contains types I, IV, and II, the second highest measurement value group will be type IV.

In addition, when bacterial cells are used to prepare a liquid sample, when the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be discriminated, whose genotype is unknown, in the sample group is compared with the fluorescence measurement results (measurement value group) in the sample group, it can be determined that the fimA gene of the periodontal disease-causing bacteria in the liquid sample is type I when the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for multiple liquid samples in the sample group is classified into the lowest measurement value group. 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 periodontal disease-causing bacteria in the liquid sample classified into the lowest measurement value group is type II.

As shown by the mottled shading in Figure 8 and the dotted shading in Figure 9, the lowest measurement value group is the measurement value group that is classified as the minimum value of fluorescence intensity among all genotypes. Similarly, the lowest measurement value group is also the measurement value group that is classified as the minimum value of the temporal change (slope) of fluorescence intensity. Therefore, if bacterial cells are used to prepare the liquid sample and the sample group contains types I, II, and IV, the lowest measurement value group will be type I. On the other hand, if a bacterial cell extract is used to prepare the liquid sample and the sample group contains types II, IV, and I, the lowest measurement value group will be type II.

Comparison of fluorescence measurement results in a sample group can be performed, for example, based on the similarity between the results obtained by the measurements. For example, the measurement values measured in a test group and the measurement values measured in a control group are compared, and when the measurement values in the test group are within a predetermined similarity range with respect to the measurement values in the control group, the fluorescence measurement results can be evaluated as being similar to each other.

The measurement values from the control group may be compared with the measurement values from the test group after having been classified (clustered) by genotype in advance. Classification by genotype can be performed using a method in which the measurement values for each genotype are classified using a predetermined threshold value, or various calculation methods such as Ward's method, group average method, longest distance method, and shortest distance method. Fluorescence measurement results can be compared using various mathematical distances such as Euclidean distance.

Alternatively, the genotype discrimination in the genotype discrimination step S30 can be performed by preparing a group of liquid samples to be discriminated, each containing a causative bacterium or bacterial cell extract (specimen) of periodontal disease of unknown genotype, and then performing an enzymatic reaction at different liquid temperatures, and then comparing the fluorescence measurement results of the liquid samples in the sample group. Note that multiple liquid samples to be discriminated are prepared that are derived from bacterial strains of the same genotype. The multiple liquid samples to be discriminated are subjected to an enzymatic reaction under substantially the same conditions for pH, the amount of causative bacterium or bacterial cell extract to be discriminated, the concentration of the fluorescently labeled substrate, etc.

The sample group can be configured to include any number of liquid samples, two or more, as the liquid samples to be distinguished, but is preferably configured to include a large number of liquid samples. Furthermore, the sample group may be configured using, in addition to the liquid samples to be distinguished, liquid samples containing periodontal disease-causing bacteria or bacterial extracts of known genotypes. By using such a sample group containing a large number of liquid samples, the behavior of the enzyme reaction in various temperature ranges can be compared for each genotype.

The liquid sample to be measured for fluorescence is preferably adjusted to a neutral pH or higher and a pH of 8.5 or lower before the fluorescence measurement and then subjected to an enzymatic reaction. The pH of the liquid sample is preferably 8.4 or lower, more preferably 8.3 or lower, even more preferably 8.2 or lower, and even more preferably 8.1 or lower. When a bacterial cell extract is used, the pH of the liquid sample is preferably 7.5 or higher, more preferably 7.8 or higher. By adjusting the pH to such a level, the enzyme activity of gingipain produced by Pg bacteria can be appropriately obtained, so that the genotype of the fimA gene of Pg bacteria can be more accurately determined. On the other hand, when bacterial cells are used, the pH of the liquid sample is preferably neutral. When bacterial cells are used, the liquid sample can be easily prepared without adjusting the pH.

It is preferable that the liquid samples to be measured for fluorescence are adjusted to different predetermined temperatures for each liquid sample in the sample group, and fluorescence measurement is performed under constant temperature control adjusted to the predetermined temperature. The liquid samples in the sample group are allocated to different temperatures within a range of 4°C to 45°C. When bacterial cells are used, it is preferable to allocate the controlled temperature of each liquid sample to at least a temperature range lower than 38 to 45°C and a temperature range higher than 38 to 45°C. When bacterial cell extracts are used, it is preferable to allocate the controlled temperature of each liquid sample to at least a temperature range lower than 4 to 15°C and a temperature range higher than 4 to 15°C, or a temperature range lower than 38 to 45°C and a temperature range higher than 38 to 45°C. When the temperatures are controlled in this way, the genotype of the fimA gene of Pg bacteria can be determined by comparing the behavior of enzyme activity at each temperature.

Specifically, the comparison of the fluorescence measurement results in the sample group can be performed by comparing the fluorescence intensity value or the amount of change in fluorescence intensity over time of the liquid sample to be distinguished, which contains the causative bacteria of periodontal disease or a bacterial extract (specimen) of unknown genotype in the sample group, with the measured values of the fluorescence intensity value or the amount of change in fluorescence intensity over time obtained for the liquid samples in the sample group other than the target to be distinguished. When making the comparison, the fluorescence intensity values or the amount of change in fluorescence intensity over time are compared among the fluorescence measurement results obtained by performing an enzyme reaction at different liquid temperatures.

When bacterial cells are used to prepare liquid samples and the temperatures of the sample group are distributed among boundary temperatures within the range of 38-45°C, if the fluorescence measurement results (measurement values) in the sample group are compared and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be identified in the sample group whose genotype is unknown is identical to or similar to the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample adjusted to a temperature range different from that of the liquid sample to be identified, it can be determined that the fimA gene of the causative bacteria of periodontal disease in the liquid sample to be identified is type I.

On the other hand, when bacterial cells are used to prepare liquid samples and the temperatures of the sample group are distributed relative to boundary temperatures within the range of 38 to 45°C, if the fluorescence measurement results (measurement values) in the sample group are compared and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be identified in the sample group whose genotype is unknown is not identical and not similar to the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample adjusted to a temperature range different from that of the liquid sample to be identified, it can be determined that the fimA gene of the bacteria causing periodontal disease in the liquid sample to be identified is type II or type IV.

In addition, when a bacterial extract is used to prepare the liquid sample, and the temperature of the sample group is allocated to boundary temperatures within the range of 4 to 15°C or 38 to 45°C, if the fluorescence measurement results (measurement values) in the sample group are compared and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be identified in the sample group whose genotype is unknown and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample adjusted to a temperature range different from that of the liquid sample to be identified are identical or similar to each other, it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample to be identified is type II.

On the other hand, when a bacterial extract is used to prepare the liquid sample, and the temperature of the sample group is allocated to boundary temperatures within the range of 4 to 15°C or 38 to 45°C, if the fluorescence measurement results (measurement values) in the sample group are compared and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample to be identified in the sample group whose genotype is unknown is not identical and not similar to the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for a liquid sample adjusted to a temperature range different from that of the liquid sample to be identified, it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample to be identified is type I or type IV.

The fluorescence measurement results of sample groups that are assigned to different temperature ranges can be compared, for example, by comparing the measured values themselves or by comparing representative values such as average values. For example, when the fluorescence measurement results of sample groups that are assigned to different temperature ranges are compared, and the difference in the fluorescence intensity values or the amount of change in fluorescence intensity over time is equal to or less than a preset threshold, it can be determined that the fluorescence measurement results are similar.

According to the discrimination method of this embodiment described above, when there is a correlation between the enzyme activity of the bacteria causing periodontal disease and the genotype of the bacteria causing periodontal disease, the genotype of the bacteria causing periodontal disease can be discriminated based on the enzyme activity determined by the fluorescence measurement method. Unlike conventional general molecular biology techniques, the enzyme activity that progresses periodontal disease is reflected in the discrimination, so that it is possible to discriminate the genotype more in line with the actual pathological condition. Furthermore, when bacteria are used to prepare the liquid sample, the sample collected from the subject's oral cavity can be used as it is for the liquid sample for fluorescence measurement, so that the genotype can be discriminated easily.

In particular, when bacterial cells are used to prepare liquid samples, the behavior of enzyme activity for each genotype changes at temperatures between 38 and 45°C, improving the accuracy of distinguishing between types II and IV and type I. Furthermore, when bacterial cell extracts are used to prepare liquid samples, the behavior of enzyme activity for each genotype changes at temperatures between 4 and 15°C or 38 and 45°C, improving the accuracy of distinguishing between types I and IV and type II. Type I Pg bacteria are more prevalent in adults with healthy periodontal tissue, while types II and IV Pg bacteria are more prevalent in adult periodontitis patients, making it possible to more accurately determine the current pathological condition of periodontal disease and the possibility of it becoming more severe.

For the preparation of liquid samples, either bacterial cells contained in dental plaque, gingival exudate, etc., or bacterial cell extracts extracted from bacterial cells can be used. It has been confirmed that the behavior of enzyme activity under temperature conditions shows different tendencies depending on the genotype and the form of the test specimen. The release property of vesicles containing enzymes, etc. from the bacterial cells and the retention by the bacterial cells may differ depending on the genotype. However, by adjusting the conditions of the enzyme reaction depending on the form of the test specimen, highly accurate discrimination can be performed.

<Fluorescence measurement device>
Next, a fluorescence measuring device according to an embodiment of the present invention will be described with reference to the drawings.

FIG. 10 is a diagram showing the configuration of a fluorescence measuring device according to an embodiment of the present invention.
As shown in FIG. 10 , the fluorescence measuring apparatus 100 of this embodiment includes a light source (irradiation means) 1, a sample holder 2, optical lenses 3 a, 3 b, a filter 4, a detection element (detection means) 5, an amplifier 6, an analog processor 7, an A/D converter 8, a control device (discrimination means) 9, a sample container 10, an input means 11, a display means 12, a pH measurement means 13, a temperature measurement means 14, and a temperature adjustment device 15.

The fluorescence measuring device 100 according to this embodiment is a fluorescence measuring device that can distinguish the genotype of bacteria that cause periodontal disease. This fluorescence measuring device 100 distinguishes which genotype, among known genetic polymorphisms, the bacteria that cause periodontal disease contained in the liquid sample Sa, or a bacterial extract (specimen) thereof, belongs to. The genotype is distinguished based on enzyme activity, the correlation of which with each genotype has been confirmed. The enzyme activity is evaluated by a fluorescence measurement method using a test agent that uses a fluorescently labeled substrate for the enzyme reaction.

The causative bacteria of periodontal disease to be identified by genotype includes Porphyromonas gingivalis (Pg bacteria), as in the identification method according to the above embodiment. The genotypes to be identified include type I (type 1), type II (type 2), type III (type 3), type IV (type 4), and type V (type 5), which are polymorphisms of the fimA gene.

As with the above-mentioned discrimination method, dental plaque, gingival exudate, etc. collected from the oral cavity of a subject can be used as is or as a suspension or the supernatant of a suspension as specimens for preparing liquid samples for fluorescence measurement to discriminate genotypes. As the liquid sample to be subjected to fluorescence measurement, a liquid sample Sa containing the causative bacteria of periodontal disease or a bacterial extract thereof and a fluorescently labeled substrate in which the substrate for the enzyme reaction induced by the causative bacteria of periodontal disease is fluorescently labeled is used.

The light source 1 is a device for generating excitation light. When an enzyme reaction occurs in the liquid sample Sa due to the bacteria that cause periodontal disease, a fluorescent chromophore dissociates from the fluorescently labeled substrate. When the liquid sample Sa is irradiated with the excitation light generated by the light source 1, the fluorescence emitted by the fluorescent chromophore is released from the liquid sample Sa.

As the light source 1, a monochromatic light source in which a specific excitation wavelength is monochromatized, such as a light emitting diode (LED) or a laser light source, is preferably used. However, as the light source 1, other light sources such as a xenon lamp, a mercury lamp, a halogen lamp, etc. may also be used together with an optical system such as a spectroscope.

The excitation light generated by the light source 1 preferably has a maximum peak at a wavelength of 350 nm or more and 380 nm or less, more preferably at a wavelength of 355 nm or more and 375 nm or less, and even more preferably at a wavelength of 360 nm or more and 370 nm or less. With such a spectrum, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, a fluorescence intensity suitable for detection can be obtained.

The sample holder 2 is installed in a measurement chamber (not shown) and supports the sample container 10 in a state in which it can be irradiated with excitation light and emit fluorescence. When measuring fluorescence, the sample container 10 containing the liquid sample Sa is fixed to the sample holder 2. The sample container 10 supported by the sample holder 2 is irradiated with excitation light from the side.

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 leaves the sample container 10, passes through the optical lens 3a, and reaches the filter 4. The filter 4 transmits only fluorescence in a specific wavelength range, excluding noise and wavelength ranges of low sensitivity. The fluorescence that has transmitted through the filter 4 passes through the optical lens 3b and enters the detection element 5, where it is converted into an electrical signal.

As the detection element 5, various detection elements such as a photodiode, a phototube, a photomultiplier tube, etc. can be used. As the filter 4, an optical filter, a dichroic mirror, etc. can be used. As the filter 4, it is preferable that the filter transmits wavelengths of 410 nm or more and 475 nm or less and blocks other wavelengths. With such characteristics, when the fluorescent chromophore constituting the fluorescent labeling substrate is AMC, the fluorescence can be detected with high sensitivity.

The fluorescent electrical signal converted by the detection element 5 is amplified by an amplifier 6, and then subjected to noise removal processing and the like by an analog processor 7 equipped with a low-pass filter and the like. The fluorescent electrical signal is then converted to a digital signal by an A/D converter 8 and input to a control device 9.

As in the above-mentioned discrimination method, the liquid sample Sa to be subjected to fluorescence measurement is preferably adjusted to a neutral pH or higher and a pH of 8.5 or lower before the fluorescence measurement and subjected to an enzymatic reaction. The pH of the liquid sample is preferably 8.4 or lower, more preferably 8.3 or lower, even more preferably 8.2 or lower, and even more preferably 8.1 or lower. When a bacterial cell extract is used, the pH of the liquid sample is preferably 7.5 or higher, more preferably 7.8 or higher. By adjusting the pH to such a level, the enzyme activity of gingipain produced by Pg bacteria can be appropriately obtained, so that the genotype of the fimA gene of Pg bacteria can be more accurately discriminated. On the other hand, when bacterial cells are used, the pH of the liquid sample is preferably neutral. When bacterial cells are used, the liquid sample Sa can be easily prepared without adjusting the pH.

The pH of the liquid sample Sa can be measured by the pH measurement means 13. For example, a glass electrode type, membrane electrode type, or other pH meter can be inserted into the sample container 10 to be used as the pH measurement means 13. The pH measurement means 13 can measure the pH of the liquid sample Sa during fluorescence measurement, so that if the pH deviates from a predetermined range, it is possible to stop the fluorescence measurement or discard inaccurate fluorescence measurement results.

As with the above-mentioned discrimination method, it is preferable to measure the fluorescence of the liquid sample Sa to be measured under constant temperature control adjusted to a predetermined temperature. The temperature of the liquid sample depends on the type of specimen and the discrimination method, but is preferably 4°C or higher and 45°C or lower.

The temperature of the liquid sample Sa can be measured by the temperature measuring means 14. For example, a thermistor, a thermocouple, a resistance temperature detector, etc. can be inserted into the sample container 10 and used as the temperature measuring means 14. The temperature measuring means 14 can monitor the temperature of the liquid sample Sa online during fluorescence measurement and feedback control the temperature control device 15.

The temperature control device 15 is a device for controlling the temperature of the liquid sample Sa in the sample container 10. The temperature control device 15 may be equipped with a PTC (Positive Temperature Coefficient) heater, a Peltier element, a constant temperature medium circulation system, etc., around the sample container 10, for example, in the sample holder 2 below or to the side. The temperature control device 15 can control the temperature of the liquid sample Sa to a temperature suitable for the enzyme reaction, allowing accurate evaluation of enzyme activity.

In the discrimination device 100 shown in FIG. 10, a cell-shaped sample container 10 is used, but a microtube can also be used as the sample container 10. Microtubes can be used for various operations such as reaction, extraction, incubation, and centrifugation, and examples of such microtubes include containers made of plastic or glass with a volume of about 1 to 2 mL.

When a microtube is used as the sample container 10, a microtube containing a liquid sample Sa with its lid removed can be attached to the sample holder 2. Excitation light can be incident on the side wall of such a microtube. Fluorescence emitted from the liquid sample Sa can be detected by being emitted above the microtube with its lid removed, rather than to the side of the sample holder 2.

In this configuration, the sidewall of the microtube acts as an optical waveguide for the excitation light, and the fluorescence is emitted above the microtube in the removed state, simplifying the optical system. In addition, the microtube used for various operations can be directly subjected to fluorescence measurement. Therefore, a small amount of liquid sample Sa is the measurement target, and a fluorescence measurement device with a simple structure can be used to perform fluorescence measurement inexpensively and easily.

FIG. 11 is a diagram showing a schematic configuration of a control device provided in the fluorescence measuring device.
As shown in FIG. 11, the control device 9 provided in the fluorescence measuring 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 memory unit 95, a display control unit 96, and a temperature control unit 97.

The control device 9 controls the operation of the fluorescence measuring device 100 and performs processing to identify the genotype of the bacteria causing periodontal disease and the bacterial extract contained in the liquid sample Sa. The control device 9 can be configured with a calculation device such as a CPU (Central Processing Unit), a storage device such as a ROM (Read Only Memory), a RAM (Random Access Memory), or a hard disk.

The control device 9 is connected to an input means 11, a fluorescence measurement unit equipped with a detection element 5, a pH measurement means 14, and a temperature measurement means 15 via an input interface (not shown). The control device 9 is also connected to a display means 12 and a temperature control device 15 via an output interface (not shown). The devices included in the control device 9 are connected to each other via a bus (not shown).

The input means 11 is a device for operating the fluorescence measuring device 100. The input means 11 can be configured with various devices such as a keyboard, a mouse, a touch pad, etc.

The display means 12 is a device that displays the results of the genotype determination. The display means 12 can be configured with various devices, such as a liquid crystal display, a plasma display, an organic EL display, etc.

The fluorescence measuring device 100 can determine the genotype of the bacteria causing periodontal disease or the bacterial extract contained in the liquid sample Sa by comparing the fluorescence intensity value or the amount of change in fluorescence intensity over time of the liquid sample containing the bacteria causing periodontal disease or the bacterial extract (specimen) of unknown genotype with a preset threshold value. When comparing, the fluorescence intensity value is compared with a threshold value corresponding to the fluorescence intensity value, or the amount of change in fluorescence intensity over time is compared with a threshold value corresponding to the amount of change in fluorescence intensity over time.

The measurement result data acquisition unit 90 acquires the measurement result data input from the fluorescence measurement unit equipped with the detection element 5. The measurement result data is data such as the fluorescence intensity of the fluorescence detected from the liquid sample Sa, and the detection time calculated from the start of the enzyme reaction. The measurement result data is output to the measurement result data processing unit 91 and the memory unit 95.

The measurement result data processing unit 91 calculates fluorescence intensity data used to distinguish genotypes based on the measurement result data. The fluorescence intensity data is data such as the fluorescence intensity value at a specified detection time and the amount of change over time (slope) of the fluorescence intensity at a specified detection time. The fluorescence intensity data may be the average value of the fluorescence intensity value in a specified detection time range, the average or maximum value of the amount of change over time (slope) of the fluorescence intensity in a specified detection time range, etc. The fluorescence intensity data is output to the measurement result data comparison unit 92 and the memory unit 95.

The measurement result data comparison unit 92 compares the fluorescence intensity data with a threshold value stored in the memory unit 95. The measurement result data comparison unit 92 determines whether the fluorescence intensity data generated for the liquid sample Sa exceeds the threshold value, and identifies the genotype of the bacteria causing periodontal disease or the bacterial extract contained in the liquid sample Sa. Data indicating the results of the identification are output to the display control unit 97.

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

The control unit 94 controls the operation of each device in the fluorescence measuring device 100, the process of determining the genotype of the bacteria causing periodontal disease, the process of displaying the results of the determination, etc., based on a predetermined program and input from the user via the input means 11.

The memory unit 95 stores programs for executing the operation of each device equipped in the fluorescence measuring device 100, the process of determining the genotype of the bacteria causing periodontal disease, the process of displaying the results of the determination, and other data, such as threshold values used to determine the genotype.

For example, a fluorescence measurement can be performed in advance using periodontal disease causative bacteria or bacterial extracts with known genotypes, and fluorescence intensity data can be obtained from the measurement result data obtained as a result of the measurement, and stored in advance in the storage unit 95. Also, a threshold value for each genotype can be set based on a large amount of fluorescence intensity data, and stored in advance in the storage unit 95.

To set the threshold, various analytical methods can be used, such as regression analysis, standard deviation classification, natural classification, and multinomial classification. The threshold can also be corrected in advance so that it is not affected by enzyme activity other than the decomposition enzyme being evaluated.

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

The temperature control unit 97 controls the temperature of the temperature control device 15. The temperature control unit 97 feedback controls the temperature control device 15 based on the temperature of the liquid sample Sa measured by the temperature measurement means 15, and maintains the temperature of the liquid sample Sa at a temperature suitable for the enzyme reaction.

FIG. 12 is a flow chart showing the flow of the discrimination method using the fluorescence measuring device.
As shown in FIG. 12, the fluorescence measuring device 100 performs fluorescence measurement on a liquid sample Sa containing a causative bacterium of periodontal disease, the genotype of which is unknown, or a bacterial extract (specimen), and can display to the user the results of the genotype determination analyzed based on the measurement data.

As shown in FIG. 12, when the fluorescence measuring device 100 is operated, the measurement conditions for fluorescence measurement are first input to the control device 9 (step S300). The measurement conditions include the type of specimen used to prepare the liquid sample, the fluorescence wavelength used in the fluorescence measurement, the waiting time/detection time, and the type of fluorescence intensity data used to distinguish the genotype, such as the fluorescence intensity value at a specified detection time and the amount of change (slope) in the fluorescence intensity over time at a specified detection time.

Next, fluorescence measurement of the liquid sample Sa containing the causative bacteria of periodontal disease, the genotype of which is unknown, or a bacterial extract (specimen) thereof is started, and excitation light is irradiated and fluorescence is detected (step S310). Then, measurement result data, which is an electrical signal of the fluorescence detected by the detection element 5, is obtained (step S320).

In step S320, if the fluorescence intensity value at a specified detection time is used to distinguish the genotype, the fluorescence intensity value at that time is collected as measurement result data in the measurement result data acquisition unit 90. If the amount of change in fluorescence intensity over time (slope) is used to distinguish the genetic system, the fluorescence intensity value over time at specified time intervals is collected as measurement result data in the measurement result data acquisition unit 90. If the average or maximum value is used, the fluorescence intensity value for a specified time range is collected.

Next, the fluorescence intensity data used to distinguish 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 a specified detection time, the average fluorescence intensity value in a specified detection time range, the amount of change over time (slope) of the fluorescence intensity at a specified detection time, the average or maximum amount of change over time (slope) of the fluorescence intensity in a specified detection time range, etc.

Next, a process is performed to determine the genotype of the periodontal disease causative bacteria or the genotype of the bacterial extract (specimen) (step S340). An image showing the results of the genotype determination is then displayed on the display means 12 by the display control unit 96 (step S350). After that, the operation of the fluorescence measuring device 100 is terminated.

The results of the genotype determination can be displayed on the display means 12, such as that the periodontal disease causative bacteria or bacterial extract with an unknown genotype contained in the liquid sample Sa belongs to any of the known genotypes, that it does not belong to any of the known genotypes, or that determination is impossible. The determination results may be displayed in any of the following ways: words, symbols, colors, etc., or together with a percentage indicating the accuracy of the determination.

FIG. 13 is a flow diagram showing the flow of a process for discriminating a genotype using a fluorescence measuring device.
As shown in FIG. 13 , the process of determining the genotype of the causative bacteria of periodontal disease or a bacterial extract of unknown genotype (step S340) is performed by comparing the fluorescence intensity data, which is the fluorescence intensity value or data on the amount of change in fluorescence intensity over time, with a threshold value stored in the memory unit 95 in the measurement result data comparison unit 92.

First, the measurement result data comparison unit 92 receives fluorescence intensity data obtained for a liquid sample containing a causative bacterium of periodontal disease, the genotype of which is unknown, or a bacterial extract (specimen) of the causative bacterium (step S341).

Next, the fluorescence intensity data of the liquid sample containing the causative bacterium of periodontal disease, the genotype of which is unknown, or its bacterial extract (specimen), i.e., the fluorescence intensity value or the amount of change in the fluorescence intensity over time, is compared with a preset first threshold value (step S342).

When the result of the comparison with the first threshold shows that the fluorescence intensity data of the liquid sample whose genotype is unknown, i.e., the fluorescence intensity value or the amount of change in fluorescence intensity over time, exceeds the first threshold (step S342: Yes), the process proceeds to step S343.

Next, the fluorescence intensity data of the liquid sample containing the causative bacterium of periodontal disease, the genotype of which is unknown, or its bacterial extract (specimen), i.e., the fluorescence intensity value or the amount of change in the fluorescence intensity over time, is compared with a second threshold value set in advance (step S343).

As the first and second thresholds, fluorescence measurements can be performed in advance using periodontal disease causative bacteria or bacterial extracts with known genotypes, and arbitrary values can be set based on the correlation between fluorescence intensity and reaction time depending on the genotype to be discriminated and the conditions of the enzyme reaction. For example, boundary values that distinguish between each genotype, such as those separating the shaded areas in Figures 8 and 9, or boundary values that distinguish between enzyme activities for each temperature range of the same genotype, can be set.

In a discrimination method for comparing fluorescence measurement results obtained by enzyme reactions at the same liquid temperature, when bacterial cells are used to prepare a liquid sample, the first threshold value can be set based on at least one of the fluorescence intensity value or the time change in fluorescence intensity of a liquid sample containing bacterial cells of a periodontal disease causative bacterium whose genotype of the fimA gene is type I, and the fluorescence intensity value or the time change in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type IV. For example, a large number of fluorescence measurement results are obtained in advance for liquid samples that are substantially the same conditions as the liquid sample Sa to be discriminated in terms of pH, temperature, amount of the periodontal disease causative bacterium to be tested, concentration of the fluorescent labeling substrate, etc., and a boundary value that is equal to or 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 can be set based on the results of the time change in fluorescence intensity. When bacterial cells are used to prepare a liquid sample, type I can be distinguished from types IV and II by the first threshold value.

On the other hand, in a discrimination method in which fluorescence measurement results obtained by enzyme reactions at the same liquid temperature are compared, when a bacterial cell extract is used to prepare a liquid sample, the first 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 a liquid sample containing a bacterial cell extract of a periodontal disease causative bacterium whose genotype of the fimA gene is type II, and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type IV. When a bacterial cell extract is used to prepare a liquid sample, the first threshold value makes it possible to distinguish type II from types IV and I.

In a discrimination method in which fluorescence measurement results obtained by enzyme reactions at the same liquid temperature are compared, when bacterial cells are used to prepare a liquid sample, the second threshold value can be set based on at least one of the fluorescence intensity value or the time change in fluorescence intensity of a liquid sample containing bacterial cells of a periodontal disease causative bacterium whose genotype of the fimA gene is type IV, and the fluorescence intensity value or the time change in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type II. For example, a large number of fluorescence measurement results are obtained in advance for liquid samples that are substantially the same conditions as the liquid sample Sa to be discriminated in terms of pH, temperature, amount of the periodontal disease causative bacterium to be tested, concentration of the fluorescent labeling substrate, etc., and a boundary value that is equal to or 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 can be set based on the results of the time change in fluorescence intensity. When bacterial cells are used to prepare a liquid sample, type II can be discriminated from types IV and I by the second threshold value.

On the other hand, in a discrimination method in which fluorescence measurement results obtained by enzyme reactions at the same liquid temperature are compared, when a bacterial cell extract is used to prepare a 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 a liquid sample containing a bacterial cell extract of a periodontal disease causative bacterium whose genotype of the fimA gene is type IV, and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type I. When a bacterial cell extract is used to prepare a liquid sample, the second threshold value makes it possible to distinguish type I from types IV and II.

In addition, in a discrimination method in which fluorescence measurement results obtained by enzymatic reactions at different liquid temperatures are compared, when bacterial cells are used to prepare a liquid sample, the first threshold value can be set based on the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium having a genotype of the fimA gene of type I or type IV is subjected to an enzymatic reaction in a temperature range lower than 38 to 45°C, and the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium of the same genotype is subjected to an enzymatic reaction in a temperature range higher than 38 to 45°C. For example, a large number of fluorescence measurement results are obtained in advance for liquid samples that are substantially the same as the liquid sample Sa to be distinguished in terms of temperature, pH, amount of bacteria causing periodontal disease, concentration of fluorescently labeled substrate, etc., and a boundary value that is equal to or greater than the maximum fluorescence measurement result measured when the enzyme reaction is performed in a temperature range higher than 38-45°C and less than the minimum fluorescence measurement result measured when the enzyme reaction is performed in a temperature range lower than 38-45°C can be set based on the results of the time change in fluorescence intensity. When bacteria are used to prepare the liquid sample, the first threshold value makes it possible to distinguish between type I and types IV and II.

On the other hand, in a discrimination method in which fluorescence measurement results obtained by enzymatic reactions at different liquid temperatures are compared, when a bacterial cell extract is used to prepare a liquid sample, the first threshold value can be set based on the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium having a genotype of the fimA gene of type I or type IV is subjected to an enzymatic reaction at a temperature range lower than 38 to 45°C, and the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium of the same genotype is subjected to an enzymatic reaction at a temperature range higher than 38 to 45°C. Alternatively, it can be set based on the fluorescence intensity value or the amount of change in fluorescence intensity over time measured when a liquid sample containing bacteria cells of periodontal disease causative bacteria with a genotype of the fimA gene of type I or type IV is subjected to an enzyme reaction in a temperature range lower than 4 to 15° C., and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured when a liquid sample containing bacteria cells of periodontal disease causative bacteria of the same genotype is subjected to an enzyme reaction in a temperature range higher than 4 to 15° C. When a bacterial cell extract is used to prepare the liquid sample, the first threshold value makes it possible to distinguish type II from types IV and I.

In addition, in a discrimination method in which fluorescence measurement results obtained by enzymatic reactions at different liquid temperatures are compared, when bacterial cells are used to prepare a liquid sample, the second threshold value can be set based on the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium having a genotype of the fimA gene of type IV or type II is subjected to an enzymatic reaction in a temperature range lower than 38 to 45°C, and the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium of the same genotype is subjected to an enzymatic reaction in a temperature range higher than 38 to 45°C. For example, a large number of fluorescence measurement results can be obtained in advance for liquid samples that are substantially the same as the liquid sample Sa to be distinguished in terms of temperature, pH, amount of bacteria causing periodontal disease, concentration of fluorescently labeled substrate, etc., and a boundary value that is equal to or greater than the maximum fluorescence measurement result measured when the enzyme reaction is performed in a temperature range higher than 38-45° C. and less than the minimum fluorescence measurement result measured when the enzyme reaction is performed in a temperature range lower than 38-45° C. can be set based on the results of the time change in fluorescence intensity. When bacteria are used to prepare the liquid sample, the second threshold value can be used to distinguish type II from types IV and I.

On the other hand, in a discrimination method in which fluorescence measurement results obtained by enzymatic reactions at different liquid temperatures are compared, when a bacterial cell extract is used to prepare a liquid sample, the second threshold value can be set based on, for example, the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium whose genotype of the fimA gene is type IV or type II is subjected to an enzymatic reaction at a temperature range lower than 38 to 45°C, and the fluorescence intensity value or the change in fluorescence intensity over time measured when a liquid sample containing bacterial cells of a periodontal disease causative bacterium of the same genotype is subjected to an enzymatic reaction at a temperature range higher than 38 to 45°C. Alternatively, it can be set based on the fluorescence intensity value or the amount of change in fluorescence intensity over time measured when a liquid sample containing bacteria cells of periodontal disease causative bacteria whose genotype of the fimA gene is type IV or type II is subjected to an enzyme reaction in a temperature range lower than 4 to 15° C., and the fluorescence intensity value or the amount of change in fluorescence intensity over time measured when a liquid sample containing bacteria cells of periodontal disease causative bacteria of the same genotype is subjected to an enzyme reaction in a temperature range higher than 4 to 15° C. When a bacterial cell extract is used to prepare the liquid sample, the second threshold value makes it possible to distinguish type I from types IV and II.

When a bacterial cell is used to prepare a liquid sample, if the result of comparison with the threshold value shows that the fluorescence intensity data of a liquid sample whose genotype is unknown, i.e., the fluorescence intensity value or the amount of change in fluorescence intensity over time, exceeds the first threshold value and is equal to or less than the second threshold value (step S342: Yes, step S343: No), it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample is a genotype other than type I or type II. In other words, if the test bacterium does not contain a genotype other than type III or type V, it can be determined that it is type IV. In addition, when a bacterial cell extract is used to prepare a liquid sample, it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample is a genotype other than type II or type I. In other words, if the test bacterium does not contain a genotype other than type III or type V, it can be determined that it is type IV.

In addition, when bacterial cells are used to prepare the liquid sample, when the result of comparison with the threshold shows that the fluorescence intensity data of the liquid sample whose genotype is unknown, i.e., the fluorescence intensity value or the time change in fluorescence intensity, exceeds the second threshold (step S342: Yes, step S343: Yes), it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample is a genotype other than type I or type IV. In other words, if the test bacterium does not contain a genotype other than type III or type V, it can be determined that it is type II. In addition, when a bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the causative bacterium of periodontal disease in the liquid sample is a genotype other than type II or type IV. In other words, if the test bacterium does not contain a genotype other than type III or type V, it can be determined that it is type I.

On the other hand, when bacterial cells are used to prepare the liquid sample, if the result of comparison with the threshold shows that the fluorescence intensity data of the liquid sample whose genotype is unknown, i.e., the fluorescence intensity value or the time change in fluorescence intensity, is equal to or less than the first threshold (step S342: No), it can be determined that the fimA gene of the periodontal disease causative bacteria in the liquid sample is a genotype other than type II or type IV. In other words, if the test bacteria does not contain any genotype other than type III or type V, it can be determined that it is type I. In addition, when a bacterial cell extract is used to prepare the liquid sample, it can be determined that the fimA gene of the periodontal disease causative bacteria in the liquid sample is a genotype other than type IV or type I. In other words, if the test bacteria does not contain any genotype other than type III or type V, it can be determined that it is type II.

According to the fluorescence measuring device of this embodiment described above, when there is a correlation between the enzyme activity of the bacteria causing periodontal disease and the genotype of the bacteria causing periodontal disease, the genotype of the bacteria causing periodontal disease can be determined based on the enzyme activity determined by the fluorescence measurement method. Unlike conventional general molecular biology techniques, the enzyme activity that progresses periodontal disease is reflected in the determination, so that the genotype can be determined in a more realistic manner according to the actual pathological condition. In addition, the sample collected from the subject's oral cavity can be used as it is as a liquid sample for fluorescence measurement, so that the genotype can be determined easily.

In particular, since the fluorescence measuring device can be equipped with a memory unit that stores the first and second threshold values, the genotype of the bacteria that cause periodontal disease can be determined stably and reproducibly regardless of the technique or operation. By simply preparing a test agent for determining the genotype of the bacteria that cause periodontal disease, a sample collected from the oral cavity of a subject with periodontal disease causative bacteria can be automatically determined, so that the current pathological state of periodontal disease and the possibility of it becoming more severe can be efficiently determined.

Although the present invention has been described above, the present invention is not limited to the above-described embodiment, and various modifications are possible within the scope of the spirit of the present invention. For example, the present invention is not necessarily limited to having all of the configurations of the above-described embodiment. It is possible to replace part of the configuration of a certain embodiment with another configuration, add part of the configuration of a certain embodiment to another form, or omit part of the configuration of a certain embodiment.

For example, the fluorescence measuring device 100 can be equipped with an appropriate optical system and signal processing system as long as it can measure the fluorescence intensity of the liquid sample Sa. The genotype can be determined not only by the first threshold value and the second threshold value, but also by other methods that compare the similarity of index values for the fluorescence intensity, such as a method similar to the determination method described above.

1. Light source (illumination means)
2 Sample holder 3a Optical lens 3b Optical lens 4 Filter 5 Detection element (detection means)
6 Amplifier 7 Analog processor 8 A/D converter 9 Control device (discrimination means)
REFERENCE SIGNS LIST 10 Sample container 11 Input means 12 Display means 13 pH measuring means 14 Temperature measuring means 15 Temperature control device 90 Measurement result data acquisition section 91 Measurement result data processing section 92 Measurement result data comparison section 93 Measurement condition data acquisition section 94 Control section 95 Storage section 96 Display control section 97 Temperature control section 100 Fluorescence measuring device

Claims (31)

A method for identifying the genotype of a causative bacterium of periodontal disease, comprising:
The causative bacterium is Porphyromonas gingivalis,
The genotype is a polymorphism of the fimA gene encoding a fimA protein,
a liquid sample containing the causative bacteria collected from the oral cavity of a subject and a fluorescently labeled reagent containing a substrate for an enzymatic reaction by the causative bacteria, the substrate being a polypeptide whose N-terminus is protected by a protecting group, and the liquid sample is subjected to an enzymatic reaction while the liquid temperature is adjusted to 4° C. or higher and 38° C. or lower, and is irradiated with excitation light;
The method for identifying the genotype is based on a time change in the fluorescence intensity of the fluorescence emitted from the liquid sample,
The liquid sample is
A liquid sample to be identified containing cells of the causative bacterium whose genotype is unknown;
and a plurality of liquid samples, each of which contains bacterial cells of the causative bacterium whose genotype is known or bacterial cells of the causative bacterium whose genotype is unknown, but does not contain a bacterial cell extract of the causative bacterium which is a supernatant obtained by centrifuging a cell suspension of the causative bacterium, and which is adjusted to the same liquid temperature as the liquid sample to be distinguished . The method according to claim 1 ,
In the genotype discrimination, when the amount of change over time in fluorescence intensity measured for the liquid sample to be discriminated is classified into the top group of measurement values among the group of measurement values of the amount of change over time in fluorescence intensity measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type II. The method according to claim 1 ,
In the genotype discrimination, when the amount of change over time in fluorescence intensity measured for the liquid sample to be discriminated is classified into the second highest measurement value group among the measurement values of the amount of change over time in fluorescence intensity measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type IV. The method according to claim 1 ,
In the genotype discrimination, when the change in fluorescence intensity over time measured for the liquid sample to be discriminated is classified into the lowest group of measurement values among the group of measurement values of the change in fluorescence intensity over time measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type I. A method for identifying the genotype of a causative bacterium of periodontal disease, comprising:
The causative bacterium is Porphyromonas gingivalis,
The genotype is a polymorphism of the fimA gene encoding a fimbrial protein,
a bacterial cell extract of the causative bacterium, which is a supernatant obtained by centrifuging a cell suspension of the causative bacterium, and a reagent in which a substrate for an enzymatic reaction by the causative bacterium is fluorescently labeled, the reagent being a polypeptide whose N-terminus is protected by a protecting group, and the liquid sample is subjected to an enzymatic reaction by adjusting the liquid temperature to 15° C. or higher and 38° C. or lower, and is irradiated with excitation light;
The method for identifying the genotype is based on a time change in the fluorescence intensity of the fluorescence emitted from the liquid sample,
The liquid sample is
A liquid sample to be identified, which contains a bacterial cell extract of the causative bacterium whose genotype is unknown;
and a plurality of liquid samples containing a bacterial body extract of the causative bacterium of a known genotype or a bacterial body extract of the causative bacterium of an unknown genotype, not containing the bacterial bodies of the causative bacterium collected from the oral cavity of a subject, and adjusted to the same liquid temperature as the liquid sample to be distinguished. The method according to claim 5 ,
In the genotype discrimination, when the amount of change over time in fluorescence intensity measured for the liquid sample to be discriminated is classified into the top group of measurement values among the group of measurement values of the amount of change over time in fluorescence intensity measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type I. The method according to claim 5 ,
In the genotype discrimination, when the amount of change over time in fluorescence intensity measured for the liquid sample to be discriminated is classified into the second highest measurement value group among the measurement values of the amount of change over time in fluorescence intensity measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type IV. The method according to claim 5 ,
In the genotype discrimination, when the change in fluorescence intensity over time measured for the liquid sample to be discriminated is classified into the lowest group of measurement values among the group of measurement values of the change in fluorescence intensity over time measured for the multiple liquid samples, the discrimination method determines that the fimA gene of the causative bacterium in the liquid sample is type II. The method according to any one of claims 1 to 8, further comprising the steps of:
The reagent is isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). The method according to any one of claims 1 to 8, further comprising the steps of:
The wavelength of the excitation light is 355 nm or more and 375 nm or less. The method according to any one of claims 1 to 8, further comprising the steps of:
The wavelength of the fluorescence is 430 nm or more and 455 nm or less. The method according to any one of claims 1 to 8, further comprising the steps of:
the liquid sample is a pH buffer solution;
The pH buffer solution contains trishydroxymethylaminomethane (Tris) as a main component. The method according to any one of claims 1 to 8, further comprising the steps of:
The method for determining whether the pH of the liquid sample is 7.0 or higher and 8.5 or lower. The method according to any one of claims 1 to 4 , further comprising the steps of:
A method for determining whether the pH of the liquid sample is neutral. A fluorescence measurement device for identifying the genotype of a causative bacterium of periodontal disease,
The causative bacterium is Porphyromonas gingivalis,
The genotype is a polymorphism of the fimA gene encoding a fimbrial protein,
an irradiation means for irradiating an excitation light onto a liquid sample containing the causative bacteria collected from the oral cavity of a subject and a fluorescently labeled reagent containing a substrate for an enzymatic reaction by the causative bacteria, the substrate being a polypeptide whose N-terminus is protected by a protecting group, the liquid temperature of which is adjusted to 4° C. or higher and 38° C. or lower;
A detection means for detecting fluorescence emitted from the liquid sample;
a discrimination means for discriminating the genotype based on a time change in the intensity of the detected fluorescence,
The liquid sample is
A liquid sample to be identified containing cells of the causative bacterium whose genotype is unknown;
a plurality of liquid samples, each of which contains bacterial cells of the causative bacterium whose genotype is known or bacterial cells of the causative bacterium whose genotype is unknown, but does not contain a bacterial cell extract of the causative bacterium, which is a supernatant obtained by centrifuging a cell suspension of the causative bacterium, and which is adjusted to the same liquid temperature as the liquid sample to be distinguished . 16. The fluorescence measuring device according to claim 15 ,
The discrimination means is
A storage unit that stores a first threshold value;
a data comparator that compares a time change in the fluorescence intensity measured for the liquid sample to be discriminated with a first threshold value set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type I when the time change in the fluorescence intensity measured for the liquid sample to be discriminated is equal to or less than the first threshold value,
The first threshold value is a value set based on at least one of the amount of change over time in fluorescence intensity measured for the liquid sample containing cells of the causative bacterium whose genotype of the fimA gene is type I, and the amount of change over time in fluorescence intensity measured for the liquid sample containing cells of the causative bacterium whose genotype of the fimA gene is type IV. 16. The fluorescence measuring device according to claim 15 ,
The discrimination means is
a storage unit that stores a first threshold value and a second threshold value;
a data comparator that compares a time change in fluorescence intensity measured for the liquid sample to be discriminated with a first threshold and a second threshold that are set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type IV when the time change in fluorescence intensity measured for the liquid sample to be discriminated exceeds the first threshold and is equal to or smaller than the second threshold,
the first threshold value is a value set based on at least one of a time change in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type I and a time change in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type IV;
The second threshold value is a value set to be greater than the first threshold value based on at least one of the amount of change over time in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type IV, and the amount of change over time in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type II. 16. The fluorescence measuring device according to claim 15 ,
The discrimination means is
A storage unit that stores a second threshold value;
a data comparator that compares a time change in the fluorescence intensity measured for the liquid sample to be discriminated with a second threshold value set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type II when the time change in the fluorescence intensity measured for the liquid sample to be discriminated exceeds the second threshold value,
The second threshold value is a value set based on at least one of the amount of change over time in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type IV, and the amount of change over time in fluorescence intensity measured for the liquid sample containing bacterial cells of the causative bacterium whose genotype of the fimA gene is type II. A fluorescence measurement device for identifying the genotype of a causative bacterium of periodontal disease,
The causative bacterium is Porphyromonas gingivalis,
The genotype is a polymorphism of the fimA gene encoding a fimbrial protein,
an irradiation means for irradiating an excitation light onto a liquid sample subjected to an enzyme reaction by adjusting a liquid temperature to 15° C. or higher and 38° C. or lower, the liquid sample containing a bacterial cell extract of the causative bacterium, which is a supernatant obtained by centrifuging a cell suspension of the causative bacterium, and a fluorescently labeled reagent containing a substrate for an enzyme reaction by the causative bacterium, the reagent being a protecting group-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide, which is a polypeptide whose N-terminus is protected by a protecting group;
A detection means for detecting fluorescence emitted from the liquid sample;
a discrimination means for discriminating the genotype based on a time change in the intensity of the detected fluorescence,
The liquid sample is
A liquid sample to be identified, which contains a bacterial cell extract of the causative bacterium whose genotype is unknown;
a plurality of liquid samples containing a bacterial body extract of the causative bacterium of a known genotype or a bacterial body extract of the causative bacterium of an unknown genotype, the liquid samples not containing the bacterial bodies of the causative bacterium collected from the oral cavity of a subject, and the liquid samples being adjusted to the same liquid temperature as the liquid sample to be distinguished. 20. The fluorescence measurement device according to claim 19 ,
The discrimination means is
A storage unit that stores a first threshold value;
a data comparator that compares a time change in the fluorescence intensity measured for the liquid sample to be discriminated with a first threshold value set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type II when the time change in the fluorescence intensity measured for the liquid sample to be discriminated is equal to or less than the first threshold value,
The first threshold value is a value set based on at least one of the amount of change over time in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type II, and the amount of change over time in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type IV. 20. The fluorescence measurement device according to claim 19 ,
The discrimination means is
a storage unit that stores a first threshold value and a second threshold value;
a data comparator that compares a time change in fluorescence intensity measured for the liquid sample to be discriminated with a first threshold and a second threshold that are set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type IV when the time change in fluorescence intensity measured for the liquid sample to be discriminated exceeds the first threshold and is equal to or smaller than the second threshold,
the first threshold value is a value set based on at least one of a time change in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type II and a time change in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type IV;
The second threshold value is a value set to be greater than the first threshold value based on at least one of the amount of change over time of fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type IV, and the amount of change over time of fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type I. 20. The fluorescence measurement device according to claim 19 ,
The discrimination means is
A storage unit that stores a second threshold value;
a data comparator that compares a time change in the fluorescence intensity measured for the liquid sample to be discriminated with a second threshold value set in advance, and determines that the fimA gene of the causative bacterium in the liquid sample is type I when the time change in the fluorescence intensity measured for the liquid sample to be discriminated exceeds the second threshold value,
The second threshold value is a value set based on at least one of the amount of change over time in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type IV, and the amount of change over time in fluorescence intensity measured for the liquid sample containing a bacterial cell extract of the causative bacterium whose genotype of the fimA gene is type I. 23. The fluorescence measuring device according to claim 15,
A fluorescence measuring device comprising a display means for displaying the result of the genotype discrimination. 23. The fluorescence measuring device according to claim 15,
A fluorescence measuring device comprising a pH measuring means for measuring the pH of the liquid sample. 23. The fluorescence measuring device according to claim 15,
A fluorescence measuring device comprising a temperature control means for adjusting the temperature of the liquid sample. 23. The fluorescence measuring device according to claim 15,
The reagent is isobutyloxycarbonyl-glycyl-glycyl-L-arginyl-4-methylcoumaryl-7-amide (iBoc-Gly-Gly-Arg-MCA). 23. The fluorescence measuring device according to claim 15,
A fluorescence measuring device, wherein the wavelength of the excitation light is 355 nm or more and 375 nm or less. 23. The fluorescence measuring device according to claim 15,
A fluorescence measuring device, wherein the wavelength of the fluorescence is 430 nm or more and 455 nm or less. 23. The fluorescence measuring device according to claim 15,
the liquid sample is a pH buffer solution;
The pH buffer solution is a fluorescence measuring device containing trishydroxymethylaminomethane (Tris) as a main component. 23. The fluorescence measuring device according to claim 15,
The pH of the liquid sample is from pH 7.0 to pH 8.5 inclusive. 19. A fluorescence measuring device according to any one of claims 15 to 18 ,
The liquid sample has a neutral pH.

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