Method for determining risk of periodontal disease and device for separating intraoral samples

Abstract

To provide a method for determining periodontal disease risk through classification into multiple graded levels across a broad spectrum, to provide an intraoral sample classification device, and to provide a method for determining disease risk.SOLUTION: The method for determining periodontal disease risk inspects the presence or absence of Pg bacteria (Porphyromonas gingivalis) in dental plaque, inspects the presence or absence of type-II Pg bacteria if Pg bacteria are detected, inspects the presence or absence of Tf bacteria (Tannerella forsythia) and the presence or absence of Td bacteria (Treponema denticola) if Pg bacteria are not detected, and classifies the dental plaque into one of five risk levels of extremely high, high, moderately high, moderate, or low. Since classification is based on the combination of the presence or absence of four types of bacteria, multi-level risk classification can be performed according to a wide range of periodontal disease risk levels.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for determining the risk of periodontal disease, an apparatus for separating oral samples, and a method for determining disease risk, and more particularly to a method for determining the risk of periodontal disease, an apparatus for separating oral samples, and a method for determining disease risk, which are used to classify and determine oral samples into multiple stages of periodontal disease risk. [Background technology]

[0002] In order to determine the risk of worsening periodontal disease, dentists generally check the depth of periodontal pockets, the presence or absence of gingival bleeding during examination, etc. Meanwhile, Patent Document 1 discloses an oral examination method including a step of measuring the bacterial load 1 of Porphyromonas gingivalis (Pg bacteria), the bacterial load 2 of Tannerella forsythia (Tf bacteria), the bacterial load 3 of Treponema denticola (Td bacteria), and the bacterial load 4 of Fusobacterium nucleatum in an oral sample collected from a subject, and a step of determining the risk of worsening of early-stage periodontal disease based on the ratio of bacterial load 4 to the total of bacterial loads 1 to 3. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7122337 Summary of the Invention [Problem to be solved by the invention]

[0004] The conventional method used by dentists was based on visible symptoms such as oral troubles, and accurate diagnosis was not possible if symptoms were not yet clearly apparent. In recent years, it has been shown that periodontal disease is related not only to oral health but also to overall health, so there is a growing emphasis on the importance of determining the risk of periodontal disease even before symptoms appear on the gums, and preventing periodontal disease early.

[0005] However, the test method described in Patent Document 1 measures the bacterial count of each type of bacteria, calculates the ratio of the sum of at least one type of specific bacteria to the total bacterial count, and determines the risk of worsening periodontal disease in the early stages of periodontal disease. Therefore, since it is necessary to measure both the specific bacteria and the total bacterial count, the measurement time may be long and the measurement accuracy is limited. Furthermore, since it is based on the ratio of the bacterial count of specific bacteria, there are limitations to its use for classifying periodontal disease risk into a wide range of multi-stage levels from low to high. Therefore, there has been a need for a method for assessing periodontal disease risk that can quickly, accurately, and efficiently classify into a wide range of multi-stage levels from low to high.

[0006] In addition, there has been a demand for a method for determining disease risk that can be efficiently and simply determined using samples collected from subjects (including not only humans but also animals), not only for periodontal disease risk but also for other disease risks.

[0007] This invention has been made to solve the above-mentioned problems, and aims to provide a method for determining periodontal disease risk that can be classified into a wide range of multi-stage grades, an apparatus for separating oral samples, and a method for determining disease risk. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, the invention described in claim 1 is a method for determining the risk of periodontal disease, which tests for the presence or absence of target bacteria in an oral sample and classifies the oral sample into one of several grades according to the degree of periodontal disease risk based on the presence or absence of the target bacteria, thereby determining the risk of periodontal disease, wherein the target bacteria include Pg bacteria (Porphyromonas gingivalis), Tf bacteria (Tannerella forsythia), and Td bacteria (Treponema denticola).

[0009] With this configuration, oral samples can be classified into multiple risk levels based on the combination of the presence or absence of at least three types of bacteria.

[0010] The invention described in claim 2 is configured as the invention described in claim 1, in which the presence or absence of Pg bacteria is tested, and if Pg bacteria is not detected, the presence or absence of Tf bacteria and Td bacteria is tested.

[0011] With this configuration, if Pg bacteria are detected, testing for the presence or absence of Tf bacteria and Td bacteria is not necessary to determine the level of periodontal disease risk.

[0012] The invention described in claim 3 is the same as the invention described in claim 1, except that the target bacteria further include Pg type II bacteria.

[0013] With this configuration, oral samples can be classified into multiple grades based on the combination of the presence or absence of at least four types of bacteria.

[0014] The invention described in claim 4 is the invention described in claim 3, in which the presence or absence of Pg bacteria is tested, and if Pg bacteria is detected, the presence or absence of Pg type II bacteria is tested, and if Pg bacteria is not detected, the presence or absence of Tf bacteria and Td bacteria is tested.

[0015] With this configuration, if Pg bacteria are detected, there is no need to test for the presence or absence of Tf bacteria and Td bacteria, and if Pg bacteria are not detected, there is no need to test for the presence or absence of Pg type II bacteria.

[0016] The invention described in claim 5 is an invention described in any one of claims 1 to 4, in which the test results for the presence or absence of target bacteria in an oral sample are compared with a rating table that associates the presence or absence of target bacteria with multiple levels of rating, and the oral sample is classified into one of multiple levels of rating.

[0017] With this configuration, oral samples can be accurately and quickly classified into multiple grades.

[0018] The invention described in claim 6 is the invention described in any one of claims 1 to 4, wherein the oral sample is saliva, dental plaque, tongue coating, oral rinse, or gingival crevicular fluid.

[0019] With this configuration, oral samples can be easily collected by anyone other than a medical professional such as a dentist or dental hygienist.

[0020] The invention described in claim 7 is an oral sample sorting device comprising a testing means for testing the presence or absence of target bacteria in an oral sample, a rating table that associates the presence or absence of target bacteria with a multi-level rating according to the degree of periodontal disease risk, and a sorting means that compares the test results for the presence or absence of target bacteria obtained by the testing means with the rating table and sorts the oral sample into one of the multi-level ratings.

[0021] With this configuration, oral samples can be accurately and quickly classified into multiple grades.

[0022] The invention described in claim 8 is the invention described in claim 7, in which the testing means comprises an amplification unit that amplifies the DNA of the target bacteria in the test solution containing the oral sample, and a detection unit that tests for the presence or absence of the amplified DNA.

[0023] With this configuration, the presence or absence of target bacteria in an oral sample can be accurately tested.

[0024] The invention described in claim 9 is the invention described in claim 8, in which the amplification unit utilizes the PCR method.

[0025] This configuration allows rapid amplification of target bacteria in oral samples.

[0026] The invention of claim 10 is the invention of claim 8, in which the detection unit utilizes a method for detecting fluorescence from a fluorescent reagent bound to the amplified DNA.

[0027] This configuration allows for easy detection of the DNA of the target bacteria.

[0028] The invention described in claim 11 is an invention described in any one of claims 7 to 10, in which the target bacteria include Pg bacteria (Porphyromonas gingivalis), Tf bacteria (Tannerella forsythia), and Td bacteria (Treponema denticola).

[0029] With this configuration, oral samples can be classified into multiple grades based on the combination of the presence or absence of at least three types of bacteria.

[0030] The invention described in claim 12 is the invention described in claim 11, in which the rating table has at least four levels: (1-0) when Pg bacteria are detected, (2-1) when Pg bacteria are not detected but both Tf and Td bacteria are detected, (2-2) when Pg bacteria are not detected but either Tf or Td bacteria are detected but the other is not, and (2-3) when Pg bacteria are not detected but neither Tf nor Td bacteria are detected.

[0031] With this configuration, oral samples can be efficiently and quickly classified into multiple stages based on the presence or absence of at least three types of bacteria.

[0032] The invention described in claim 13 is the invention described in claim 11, wherein the target bacteria further include Pg type II bacteria.

[0033] With this configuration, oral samples are classified into multiple grades based on the combination of the presence or absence of at least four types of bacteria.

[0034] The invention described in claim 14 is the invention described in claim 13, in which the rating table has at least five levels: (1-1) when Pg bacteria are detected but Pg type II bacteria are detected, (1-2) when Pg bacteria are detected but Pg type II bacteria are not detected, (2-1) when Pg bacteria are not detected but both Tf bacteria and Td bacteria are detected, (2-2) when Pg bacteria are not detected but either Tf bacteria or Td bacteria are detected but the other is not, and (2-3) when Pg bacteria are not detected but neither Tf bacteria nor Td bacteria are detected.

[0035] With this configuration, oral samples can be efficiently and quickly classified into multiple stages based on the presence or absence of at least five types of bacteria.

[0036] The invention of claim 15 is the invention of claim 7, further comprising an output unit that outputs information on the grade of the oral sample separated by the separation means to the outside of the device.

[0037] This configuration makes it easier to confirm the grade of the separated oral sample.

[0038] The invention described in claim 16 is a method for determining the risk of a disease, comprising: an inspection step of inspecting the presence or absence of multiple types of target bacteria in a sample; and a determination step of classifying the sample into one of multiple grades corresponding to the degree of target disease risk prepared in advance, based on the combination of the presence or absence of the multiple types of target bacteria, to determine the risk of the target disease.

[0039] With this configuration, the risk of a target disease can be determined simply by testing for the presence or absence of multiple types of target bacteria.

[0040] The invention described in claim 17 is the invention described in claim 16, in which a level of importance is set in advance for each of multiple types of target bacteria, and in the judgment process, the test results for the presence or absence of target bacteria set to a relatively high level of importance are weighted and evaluated.

[0041] This configuration allows for more accurate assessment of disease risk.

[0042] The invention described in claim 18 is the invention described in claim 17, in which, in the testing process, based on the test results for the presence or absence of the target bacteria for which the importance is set relatively high, the testing of the target bacteria for which the importance is set relatively low is either omitted or tested.

[0043] This configuration allows for a more rapid assessment of disease risk. [Effects of the Invention]

[0044] As explained above, the invention described in claim 1 can classify oral samples into multiple risk levels based on the combination of the presence or absence of at least three types of bacteria, thereby enabling efficient and easy assessment by classifying into multiple levels corresponding to a wide range of periodontal disease risk levels.

[0045] In addition to the effect of the invention of claim 1, the invention of claim 2 makes it possible to efficiently separate oral samples, since when Pg bacteria are detected, testing for the presence or absence of Tf bacteria and Td bacteria is not necessary to distinguish the degree of periodontal disease risk.

[0046] The invention described in claim 3 has the effect of the invention described in claim 1, and in addition, can classify oral samples into multiple grades based on the combination of the presence or absence of at least four types of bacteria, thereby enabling classification into even more grades according to a wider range of periodontal disease risk levels.

[0047] In addition to the effects of the invention of claim 3, the invention of claim 4 makes it unnecessary to test for the presence or absence of Tf bacteria and Td bacteria when Pg bacteria are detected, and unnecessary to test for the presence or absence of Pg type II bacteria when Pg bacteria are not detected, thereby enabling efficient separation of oral samples.

[0048] The invention of claim 5 has the effect of the invention of any one of claims 1 to 4, and in addition, can accurately and quickly separate oral samples into multiple grades, making it possible to separate samples in clinical settings.

[0049] The invention of claim 6 has the effect of the invention of any one of claims 1 to 4, and in addition, oral samples can be easily collected by anyone other than a medical professional such as a dentist or dental hygienist, and therefore can be easily separated.

[0050] The invention described in claim 7 can accurately and quickly separate oral samples into multiple grades, and can therefore be used in clinical settings.

[0051] The invention of claim 8 has the effect of the invention of claim 7, and in addition, can accurately test for the presence or absence of target bacteria in oral samples, thereby enabling accurate classification of oral samples.

[0052] The invention described in claim 9 has the effect of the invention described in claim 8, and in addition, can rapidly amplify target bacteria in oral samples, so that even trace amounts of target bacteria can be detected and oral samples can be accurately and quickly distinguished.

[0053] The invention of claim 10 has the same effect as the invention of claim 8, and in addition, allows the DNA of the target bacteria to be easily detected, so that oral samples can be easily separated.

[0054] The invention described in claim 11 has the effect of any of the inventions described in claims 7 to 10, and in addition, classifies oral samples into multiple grades based on the combination of the presence or absence of at least three types of bacteria, making it possible to classify oral samples into multiple grades according to the degree of periodontal disease risk over a wide range.

[0055] The invention of claim 12 is useful in clinical settings because, in addition to the effects of the invention of claim 11, it can efficiently and quickly differentiate oral samples in multiple stages based on the combination of the presence or absence of at least three types of bacteria.

[0056] The invention described in claim 13, in addition to the effect of the invention described in claim 11, classifies oral samples into multiple grades based on the combination of the presence or absence of at least four types of bacteria, making it possible to classify oral samples into even more grades according to the degree of periodontal disease risk over an even wider range.

[0057] The invention described in claim 14 is useful in clinical settings because, in addition to the effects of the invention described in claim 13, it can efficiently and quickly differentiate oral samples into multiple stages based on the combination of the presence or absence of at least five types of bacteria.

[0058] The invention of claim 15 has the same effect as the invention of claim 7, and further improves convenience by making it easier to check the grade of the separated oral samples.

[0059] The invention described in claim 16 can determine the risk of a target disease simply by testing for the presence or absence of multiple types of target bacteria, allowing for efficient and simple determination.

[0060] The invention of claim 17 has the same effect as the invention of claim 16, and in addition, can more accurately determine disease risk, thereby improving usefulness.

[0061] The invention of claim 18 has the same effect as the invention of claim 17, and in addition, can determine disease risk more quickly, thereby further improving its usefulness. [Brief explanation of the drawings]

[0062] [Figure 1] FIG. 4 is a process diagram showing the steps of a method for determining the risk of periodontal disease according to a second embodiment of the present invention. [Figure 2] FIG. 10 is a process diagram showing the steps of a method for determining the risk of periodontal disease according to a fourth embodiment of the present invention. [Figure 3] FIG. 10 is a schematic diagram showing the configuration of an oral cavity sample separation device according to a fifth embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0063] A method for determining the risk of periodontal disease (intraoral examination method) according to a first embodiment of the present invention will be described.

[0064] Table 1 is a rating table according to the first embodiment that associates the presence or absence of target bacteria in dental plaque, which is an oral sample, with a four-level rating according to the degree of periodontal disease risk. The periodontal disease risk includes the risk of developing periodontal disease in the subject from whom the oral sample was collected (possibility of developing periodontal disease, severity of symptoms at the time of onset) and the risk of the periodontal disease worsening (possibility of worsening, degree of worsening). The rating of dental plaque according to the degree of periodontal disease risk serves as an indicator for detailed examination, diagnosis, preventive measures, treatment, etc. of periodontal disease.

[0065] [Table 1] Dental plaque collected from subjects is classified into one of several grades according to the degree of periodontal disease risk based on the presence or absence of three target bacteria (Pg, Tf, and Td) in the plaque. Specifically, plaque is classified into four grades: "High," which indicates a high degree of periodontal disease risk; "Medium+," which indicates a moderately high degree of periodontal disease; "Medium," which indicates a moderate degree of periodontal disease risk; and "Low," which indicates a low degree of periodontal disease risk. The details of the periodontal disease risk corresponding to each grade are described below.

[0066] Referring to Table 1, if Pg bacteria are detected (corresponding to the "Present" case in Table 1, meaning that the test for the presence (presence) of Pg bacteria reveals a Pg count of 100 or more bacteria per μL, confirming their presence. The bacterial count is based on the count measured after dilution of 400x for dental plaque, 20x for saliva, 20x for oral rinse, and 20x for gingival crevicular fluid. For other samples, the dilution ratio should be set as appropriate, following the same procedure: 400x for solid or paste samples, and 20x for liquid samples. The same rule applies hereafter regardless of the type of bacteria.), regardless of the presence or absence of Tf and Td bacteria. This is because Pg bacteria are the oral bacteria most closely associated with the risk of periodontal disease.

[0067] On the other hand, if Pg bacteria are not detected (corresponding to the case of "absent" in Table 1, which means that as a result of testing for the presence (presence) of Pg bacteria, the number of Pg bacteria is less than 100 / μL and the presence of Pg bacteria is not confirmed; hereinafter, this applies regardless of the type of bacteria), the plaque is graded as less than high, and is further classified into three levels depending on the presence or absence of Tf and Td bacteria. That is, if both Tf and Td bacteria are detected, the plaque is graded as medium+. Also, if either Tf or Td bacteria are detected but the other is not, the plaque is graded as medium. Furthermore, if neither Tf nor Td bacteria are detected, the plaque is graded as low.

[0068] In the first embodiment of the present invention, dental plaque is classified according to the combination of the presence or absence of at least three types of bacteria, and therefore can be classified into multiple grades according to a wide range of levels of periodontal disease risk.

[0069] Fig. 1 is a process diagram showing the steps of a method for determining the risk of periodontal disease (a method for distinguishing dental plaque) according to a second embodiment of the present invention. Table 2 is a rating table according to the second embodiment, which associates the presence or absence of target bacteria in dental plaque with a four-level rating according to the degree of periodontal disease risk.

[0070] [Table 2] Referring to Figure 1 and Table 2, in the second embodiment, similar to the first embodiment, dental plaque is classified into one of four grades: "high," "medium+," "medium," and "low" depending on the presence or absence of three types of target bacteria (Pg bacteria, Tf bacteria, and Td bacteria) in the dental plaque.

[0071] Referring to Fig. 1, the presence or absence of Pg bacteria in dental plaque is examined in Pg bacteria examination step 1. If Pg bacteria are detected, the dental plaque is graded as high, and examination of the presence or absence of Tf bacteria and Td bacteria is not necessary.

[0072] On the other hand, if Pg bacteria are not detected in Pg bacteria testing step 1, the presence or absence of Tf bacteria in the plaque is tested in Tf bacteria testing step 2, and after Tf bacteria testing step 2, the presence or absence of Td bacteria in the plaque is tested in Td bacteria testing step 3.

[0073] If both Tf and Td bacteria are detected, the plaque is graded as medium+. If Tf bacteria are detected but not Td bacteria, or if Tf bacteria are not detected but Td bacteria are, the plaque is graded as medium. If neither Tf nor Td bacteria are detected, the plaque is graded as low.

[0074] In the second embodiment, dental plaque is classified according to the presence or absence of at least three types of bacteria, allowing for classification into multiple grades corresponding to a wide range of periodontal disease risk levels. Furthermore, if Pg bacteria are detected, the dental plaque is classified as high, eliminating the need to test for the presence or absence of Tf and Td bacteria, allowing for efficient classification into multiple grades corresponding to a wide range of periodontal disease risk levels.

[0075] Table 3 is a rating table according to the third embodiment, which associates the presence or absence of target bacteria in dental plaque with a five-level rating according to the degree of periodontal disease risk.

[0076] [Table 3] Plaque collected from subjects is classified into one of five grades according to the degree of periodontal disease risk, based on the presence or absence of four target bacteria (Pg, Pg type II, Tf, and Td) in the plaque.

[0077] Specifically, dental plaque is classified into five grades: "High+," which means the degree of periodontal disease risk is extremely high, "High," which means high, "Medium+," which means moderately high, "Medium," which means moderate, and "Low," which means low. The details of periodontal disease risk corresponding to each grade are as follows: High+: Periodontal pocket depth 6mm or more, regardless of whether or not there is gingival bleeding High: Periodontal pocket depth 4mm to less than 6mm, gingival bleeding Medium+: Periodontal pocket depth 4mm to less than 6mm, no gingival bleeding Medium: Periodontal pocket depth less than 4mm, gingival bleeding Low: Periodontal pockets less than 4mm, no gingival bleeding If Pg bacteria are detected, the plaque is graded as high or higher (including both high and high+), regardless of whether Tf and Td bacteria are present. This is because Pg bacteria are most closely associated with the risk of periodontal disease among all bacteria present in the oral cavity.

[0078] If Pg type II is detected, the plaque is graded as High+, regardless of the presence or absence of Tf and Td. Pg bacteria exist in six distinct genotypes, depending on the nucleic acid sequence structure of fimA, which encodes the fimbrillin protein. They are classified into six different fimbriae types. The pathogenesis of periodontal disease varies depending on the fimbriae type of Pg. Among these, type II fimbriae is highly pathogenic and has the greatest association with periodontal disease risk among Pg bacteria. Therefore, its presence or absence is weighted and evaluated accordingly. By testing for Pg type II independently of the presence or absence of Pg, plaque graded as High or higher can be further classified into two levels: High and High+.

[0079] Table 4 shows the odds ratio for the development of periodontal disease for each type of Pg bacteria (source: "Dr. Amano's Periodontal Disease Picture Book: The Story of the Biofilm Principality"). Referring to Table 4, it can be seen that Pg bacteria have a high odds ratio for the development of periodontal disease, although the degree varies depending on the type, and that Pg type II in particular has an extremely high odds ratio for the development of periodontal disease.

[0080] [Table 4] On the other hand, if Pg bacteria are not detected, the plaque is graded as less than high, as in the first embodiment, and is further classified into three grades depending on the presence or absence of Tf and Td bacteria. That is, the plaque is graded as Medium+ when both Tf and Td bacteria are detected, as Medium when either Tf or Td bacteria are detected but the other is not, and as Low when neither Tf nor Td bacteria are detected.

[0081] In the third embodiment, dental plaque is classified according to a combination of the presence or absence of at least four types of bacteria, and therefore can be classified into even more grades according to a wider range of levels of periodontal disease risk.

[0082] 2 is a process diagram showing the steps of a method for determining the risk of periodontal disease (a method for distinguishing dental plaque) according to a fourth embodiment of the present invention. Table 5 is a rating table according to the fourth embodiment, which associates the presence or absence of target bacteria in dental plaque with a five-level rating according to the degree of periodontal disease risk.

[0083] [Table 5] Referring to Figure 2 and Table 5, dental plaque is classified into one of five grades: "High+", "High", "Medium+", "Medium", and "Low" depending on the presence or absence of four types of target bacteria (Pg, Pg type II, Tf, and Td) in the plaque.

[0084] Referring to Figure 2, in Pg bacteria testing step 1, the presence or absence of Pg bacteria in dental plaque is tested. If Pg bacteria are detected, the dental plaque is graded as high or higher, and testing for the presence or absence of Tf and Td bacteria is not necessary. In Pg type II testing step 5, the presence or absence of Pg type II bacteria in dental plaque is tested. If Pg type II bacteria is detected, the dental plaque is graded as high+. If Pg bacteria are detected but Pg type II bacteria is not detected, the dental plaque is graded as high.

[0085] Pg bacteria exist in at least the types listed in Table 4. Therefore, if at least one of the types listed in Table 4 is detected in Pg testing step 1, Pg bacteria have been detected. On the other hand, if Pg bacteria are not detected, it means that none of the types of Pg bacteria, including Pg type II, have been detected. In other words, when testing for the presence or absence of Pg type II after Pg testing step 1, if Pg bacteria are not detected in Pg testing step 1, it can be said that testing for the presence or absence of Pg type II is not necessary.

[0086] On the other hand, if Pg bacteria are not detected in Pg bacteria testing step 1, the presence or absence of Tf bacteria in the plaque is tested in Tf bacteria testing step 2, and after Tf bacteria testing step 2, the presence or absence of Td bacteria in the plaque is tested in Td bacteria testing steps 3 and 4.

[0087] The plaque grade was rated as medium+ if both Tf and Td bacteria were detected, as medium if Tf bacteria were detected but not Td bacteria, or if Tf bacteria were not detected but Td bacteria were detected, and as low if neither Tf nor Td bacteria were detected.

[0088] In the fourth embodiment, if Pg bacteria are detected, there is no need to test for the presence or absence of Tf bacteria and Td bacteria, and if Pg bacteria are not detected, there is no need to test for the presence or absence of Pg type II bacteria, so dental plaque can be efficiently classified. Furthermore, by comparing the test results for the presence or absence of bacteria in dental plaque with a classification table, it is possible to accurately and quickly classify the bacteria into multiple grades, making classification possible in clinical settings. Furthermore, dental plaque, which is an oral sample, can be easily collected, making it easy to classify. For these reasons, it is useful in clinical settings.

[0089] FIG. 3 is a schematic diagram showing the configuration of an oral cavity sample separation device according to a fifth embodiment of the present invention.

[0090] Referring to FIG. 3, the sorting device 10 includes an inspection means 20, a classification table (not shown), and a sorting means 30.

[0091] The testing means 20 is a means for testing the presence or absence of target bacteria in dental plaque, which is an oral sample, and includes an amplification unit 21, a detection unit 22, and a control unit (not shown). The amplification unit 21 amplifies the DNA of target bacteria in a test solution containing dental plaque by PCR, and progresses the PCR by heating and cooling the test solution. The detection unit 22 tests the presence or absence of amplified DNA in the test solution after PCR has been completed in the amplification unit 21 by optical detection, and utilizes a method of detecting fluorescence from a fluorescent reagent bound to the amplified DNA. The control unit controls the operations of the amplification unit 21 and the detection unit 22.

[0092] The amplification unit 21 is mainly composed of a container holder that holds the test container, a support that supports the container holder, a plate-shaped first heater, a ring-plate-shaped heating plate fixed above the first heater, a plate-shaped second heater, and a base that fixes the first heater and second heater to their upper surfaces.

[0093] The test container contains each component of the test solution (plaque sample, mixed solution, fluorescent reagent, etc.) separated by a paraffin partition, and when the paraffin partition is heated and melted in the amplification section 21, the components are mixed to form the test solution, which is then stored in the lower part of the test container.

[0094] The container holder holds the test container so that the bottom surface of the test container is horizontal with the surfaces of the first heater and the second heater. The support part allows the container holder to slide from the first heater to the second heater or vice versa, thereby allowing the test container to be freely moved above the first heater, above the second heater, or to the detection part 22.

[0095] The first heater is configured so that its temperature can be freely adjusted by the control unit, and has a recess on its upper surface into which the lower part of the cuvette can be fitted, and the surface of the recess is set to a high temperature, for example, 120°C, by the control unit.

[0096] The heating plate has a through-hole in the center through which the cuvette can be inserted, and the inner surface of the through-hole is heated by heat conduction from the first heater to, for example, 80°C.

[0097] The second heater has a recess on its upper surface into which the lower part of the test vessel can fit, and is the same as the first heater described above except for the temperature, and the surface of the recess is set by the control unit to a lower temperature than the first heater, for example, 50°C.

[0098] The base is controlled by the control unit to slide freely in the direction of the recesses of the first heater and the second heater or in the opposite direction, thereby allowing the first heater and the second heater to be raised or lowered freely.

[0099] First, the base rises, and the lower part of the test container passes through the through-hole of the rising heating plate and fits into the recess of the first heater. The test container is heated by the heating plate, the paraffin insulating the middle and lower parts of the test container melts, and the plaque sample stored in the upper part of the test container and the mixed solution stored in the middle part are released into the lower part, mixed together to become the test solution, which is stored in the lower part of the test container and heated by the contact (first contact) between the surface of the lower part of the test container and the surface of the recess of the first heater.

[0100] When the heating by the first contact for a predetermined time is completed, the base descends and the first contact is released. The support then moves the vessel holder horizontally so that the test vessel is above the second heater. As with the first contact, the base rises, causing the bottom of the test vessel to fit into the recess of the second heater, resulting in contact (second contact) between the surface of the bottom of the test vessel and the surface of the recess of the second heater. This second contact cools the test solution to, for example, 65°C, and annealing is performed.

[0101] When cooling by the second contact for a predetermined time is completed, the base descends, the support unit moves the container holder horizontally, and the test container moves above the first heater. PCR progresses as the first contact and the second contact are alternately repeated in this manner. Then, after the first contact and the second contact are repeated a predetermined number of times and PCR is completed, the support unit moves the container holder, and the test container moves to the detection unit 22.

[0102] The detection unit 22 is mainly composed of a light-emitting element and a light-receiving element. The light-emitting element and the light-receiving element are arranged to face each other on a horizontal coaxial line, and the cuvette is arranged so that the line passes through the bottom of the cuvette. The light-emitting element is an LED lamp with an effective wavelength of 450 nm to 570 nm. The light-receiving element is a photodiode with an effective wavelength of 300 nm to 820 nm.

[0103] In the detection unit 22, a light-emitting element emits excitation light toward the test solution contained in the lower part of the test container. The amplified DNA in the test solution binds to the fluorescent reagent contained in the test solution, reacts to the excitation light, fluoresces, and the fluorescence that passes through the lower part of the test container made of a transparent material is received by a light-receiving element.

[0104] Referring to Tables 1 to 3 and 5, the rating tables associate the presence or absence of target bacteria with a multi-level rating according to the degree of periodontal disease risk. Depending on the purpose of the test, at least one rating table can be selected in advance from the rating tables shown in the first embodiment (Table 1), second embodiment (Table 2), third embodiment (Table 3) and fourth embodiment (Table 5) of the present invention.

[0105] The classification means 30 compares the test results for the presence or absence of target bacteria obtained by the test means 20 with a classification table and classifies the dental plaque into one of a plurality of grades. The classification means 30 can be an electronic calculator (computer) equipped with a processing device (semiconductor chip) that integrates a control device and a calculation device, a memory device, an input device, an output device, etc. The classification means 30 can also serve as the control unit of the test means 20.

[0106] In the fifth embodiment, dental plaque can be accurately and quickly classified into multiple grades, making it suitable for use in clinical settings. Furthermore, the presence or absence of target bacteria in dental plaque can be accurately tested, allowing for accurate classification of dental plaque. Furthermore, target bacteria in dental plaque can be rapidly amplified, allowing even trace amounts of target bacteria to be detected, enabling accurate and rapid classification of dental plaque. Furthermore, the DNA of target bacteria can be easily detected, allowing for easy classification of dental plaque. Furthermore, dental plaque can be classified into multiple grades based on the combination of the presence or absence of at least three types of bacteria, allowing for classification into multiple grades corresponding to a wide range of periodontal disease risk levels. Furthermore, dental plaque can be classified into multiple grades based on the combination of the presence or absence of at least four types of bacteria, allowing for even more extensive classification based on an even wider range of periodontal disease risk levels.

[0107] The oral sample sorting device according to the fifth embodiment may also include an output unit (not shown). The output unit outputs information on the grade of the oral sample sorted by the sorting means to an external device. More specifically, the output unit may display the information on a monitor installed in the sorting device, print it out on paper, or display it on an app or software on a smartphone or personal computer connected to the sorting device. This configuration makes it easier to check the grade of the sorted oral sample, further improving convenience. Furthermore, the output unit may output the periodontal disease risk assessment results linked to the oral sample grade information, future predicted symptoms based on the assessed periodontal disease risk, and information on preventive measures and treatments to reduce periodontal disease risk. While the sorting device of the present invention does not perform diagnostic procedures, a dentist or other professional may diagnose the condition and output the above information from the output unit. Furthermore, such output is not limited to dentists selecting the appropriate content from various content previously stored in the sorting device, but can also include additions or replacements of content entered by dentists, dental hygienists, or other medical professionals at the site where the examination is performed. [Example]

[0108] The 104 subjects were examined for periodontal pocket depth (PPD) and the presence or absence of gingival bleeding (BOP) during the examination, and dental plaque samples were collected and examined for the presence or absence of Pg bacteria, Pg type II bacteria, Tf bacteria, and Td bacteria.The presence or absence of each target bacteria was compared with the grading table in Table 3, and the dental plaque of each subject was classified into five levels. [1] Testing for the presence or absence of target bacteria The presence or absence of each target bacterium in each dental plaque was examined using a bacterial testing device (Orcoa, manufactured by Yamato Eslon Co., Ltd.) with PCR and fluorescence detection functions. That is, using the bacterial testing device, the DNA of each target bacterium in the test solution mixed with each dental plaque was amplified by PCR, and the presence or absence of each target bacterium was examined by fluorescence detection. (1) Preparation of test solution 1.0 μL of dental plaque was collected from the oral cavity of each subject using a collection tool, and 1.0 μL of phosphate buffered saline (PBS) (Sigma-Aldrich) and 9.0 μL of ultrapure water were added and mixed to prepare a dental plaque sample.

[0109] Next, a test container with a paraffin partition inside was used, and the test container containing 0.28 μL of plaque sample, 8.0 μL of mixed solution, and 2.0 μL of fluorescent reagent (SYBR (registered trademark) Green II, manufactured by Takara Bio Inc.) separated by a paraffin partition was held in the container holder of the bacteria testing device.

[0110] The mixed solution used was prepared by mixing the following reagents in the following proportions: ·Water (ultra pure water) 15.0μL Buffer solution (10x TE buffer, 30 mM MgCl2 solution: Takara Bio Inc.) 2.5 μL 2.5 mM dNTP (Takara Bio Inc., product name: dNTP Mixture) 2.0 μL 10μM primer I for each periodontal pathogen 1.0μL 10μM primer II for each periodontal pathogen 1.0μL DNA polymerase (Takara Bio Inc., product name: SpeedSTAR HS DNA Polymerase) 0.126 μL Primers I for each periodontal disease bacteria and primers II for each periodontal disease bacteria were mixed according to the target bacteria to be tested (primers for each target bacteria) to prepare a test solution used to test for the presence or absence of each target bacteria.

[0111] The following commercially available primers can be used for each target bacterium. ·For Pg bacteria Primer I: Life Technologies, product name: PG Primer I Primer II: Life Technologies, product name: PG Primer II ·For Tf bacteria Primer I: Life Technologies, product name: TF Primer I Primer II: Life Technologies, product name: TF Primer II For Td bacteria Primer I: Life Technologies, product name: TD Primer I Primer II: Life Technologies, product name: TD Primer II ·For Pg bacteria type II Primer I: Life Technologies, product name: PGII type primer I Primer II: Life Technologies, product name: PGII type primer II The sequences of the primers for each of the target bacteria mentioned above are as follows:

[0112] [Table 6] Pg type II Primer I: ACA ACT ATA CTT ATG ACA ATG G Primer II: AAC CCC GCT CCC TGT ATT CCG A (2) PCR treatment The bacterial testing device was set at room temperature (26°C), a test container was placed in the container holder, and the test container was preheated using the heating plate of the amplification unit 21 (temperature 80°C) to melt the paraffin partition.The thermal cycle in which the lower part of the test container was brought into contact (heated) with a first heater (temperature 120°C) for a predetermined time, and then brought into contact (cooled) with a second heater (temperature 50°C) for a predetermined time was repeated 50 times. (3) Testing for the presence or absence of each target bacteria The test solution and control solution after PCR treatment are each irradiated with light, and the light emitted from the test solution and control solution after irradiation is received by a detection element, and the voltage generated by the detection element is measured. The control solution was the same as the test solution, except that it was mixed with dental plaque that was confirmed to be free of the target bacteria.

[0113] If the target bacteria are present in the dental plaque mixed with the test solution, the DNA of the target bacteria in the test solution is amplified by PCR and binds to a fluorescent reagent (SYBR (registered trademark) Green II) in the test solution. When the test solution is irradiated with LED light, the fluorescent reagent bound to the DNA of the target bacteria absorbs the LED light and emits green light. Therefore, the presence or absence of the target bacteria in the dental plaque can be confirmed by detecting the green light emitted from the test solution with a test element.

[0114] For light irradiation, an LED lamp (manufactured by Nichia Corporation, emission center wavelength: 495 nm) was used, and as a detection element, a photodiode (manufactured by Hamamatsu Photonics K.K., spectral sensitivity characteristics: 300 nm to 850 nm) was used.

[0115] When the target bacteria are present in the test solution, the voltage generated when the photodiode receives the light emitted from the test solution is significantly greater than the voltage generated when it receives the light emitted from the control solution.Therefore, the difference in the amount of green light (central emission wavelength: 520 nm) emitted from the test solution, which comes from the fluorescence of the fluorescent reagent bound to the DNA of the target bacteria, was confirmed as a change in the voltage of the electrical signal output from the photodiode.

[0116] To measure the amount of target bacteria in dental plaque, it is necessary to measure and quantify the intensity of green light received by the detection element. In contrast, the presence or absence of each target bacterium in dental plaque can be tested, for example, by checking the fluorescence (green light) from the test solution after PCR, as described above. That is, the presence or absence of target bacteria can be tested by setting a threshold value (threshold value of the intensity of green light received by the detection element) for determining the presence or absence of the target bacterium. Since precise measurement (quantification) of the bacterial amount is not required, this test is quicker and easier than other tests that require precise measurement of the bacterial amount. [2] Plaque separation The relationship between the classification results of each plaque grade in this example and the examination results of PPD (periodontal pocket depth) and BOP (bleeding when measuring pockets in a periodontal disease test) is shown in Tables 7 and 8.

[0117] Table 7 shows the number of plaques classified into each grade, categorized by the examination results of the subjects from whom the plaque was collected, showing whether they had PPD or BOP.

[0118] From Table 7, it can be seen that the plaque classification results (grade) obtained in this example have a significant causal relationship with the examination items (presence or absence of BOP and PPD) that are considered to be closely related to periodontal disease risk in dental practice.

[0119] [Table 7] Table 8 shows the percentage of plaque that falls within the range (within the double border in Table 8) where each grade and the subject's examination results (presence or absence of PPD and BOP) are roughly consistent, based on the number of plaques classified by the plaque grades (classification results) in Table 7 and the presence or absence of PPD and BOP for each grade of plaque.

[0120] [Table 8] Referring to Table 8, of the plaque classified as "high," the proportion of plaque within the range generally consistent with the subject's examination results (PPD 4mm or more with or without BOP) was 91%, of the plaque classified as "medium+," the proportion of plaque within the range generally consistent with the subject's examination results (PPD less than 6mm with BOP or PPD less than 6mm and 4mm or more without BOP) was 50%, of the plaque classified as "medium," the proportion of plaque within the range generally consistent with the subject's examination results (PPD less than 4mm with BOP or PPD less than 6mm without BOP) was 29%, and of the plaque classified as "low," the proportion of plaque within the range generally consistent with the subject's examination results (PPD less than 4mm with or without BOP) was 58%, indicating generally high consistency within a range with a sufficient population size.

[0121] For example, out of 16 plaques in which the subject's examination results were in the good range (no BOP and PPD less than 4mm), three plaques were classified as "high" or "medium+." This suggests that even if there is a latent risk of periodontal disease that cannot be confirmed by visual examination of the subject, the presence or absence of target bacteria in the plaque can be used to classify the plaque into grades according to the degree of periodontal disease risk, including the latent risk of periodontal disease. The same applies to other ranges.

[0122] Furthermore, although this example corresponds to the first embodiment, similar separation results can be obtained with fewer tests in the second embodiment, in which the presence or absence of Pg bacteria makes it unnecessary to test for the presence or absence of Tf and Td bacteria and therefore is not performed.

[0123] Furthermore, in the third and fourth embodiments, which target Pg type II bacteria and classify "high" into "high+" and "high," the consistency between the plaque grade and the subject's examination results can be evaluated in the same way from Tables 7 and 8, as in the first and second embodiments.

[0124] In the above embodiments, dental plaque is classified into multiple grades as an oral sample, but oral samples other than dental plaque can also be classified into multiple grades according to the degree of periodontal disease risk, such as saliva, oral rinse, or gingival crevicular fluid.

[0125] Furthermore, in the first and second embodiments, the target bacteria include three types of bacteria (Pg, Tf, and Td), and in the third and fourth embodiments, the target bacteria include four types of bacteria (Pg, Pg type II, Tf, and Td). However, other bacteria may also be included. Examples of other bacteria include Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Aggregatibacter actinomycetemcomitans, Filifactor alocis, and Porphyromonas gulae. This configuration allows oral samples to be classified into multiple grades more accurately.

[0126] Furthermore, in the second and fourth embodiments described above, the Td bacterial testing step is performed after the Tf bacterial testing step, but the Tf bacterial testing step may be performed after the Td bacterial testing step, or the Tf bacterial testing step and the Td bacterial testing step may be performed simultaneously or in parallel. By configuring the Tf bacterial testing step and the Td bacterial testing step to be performed simultaneously or in parallel, dental plaque can be separated even more quickly.

[0127] Furthermore, in the fourth embodiment, the Pg type II testing step is performed when Pg bacteria are detected in the Pg type II testing step, but the Pg type II testing step may also be performed when Pg type II is not detected in the Pg type II testing step. By configuring in this way, if Pg type II is detected in the Pg type II testing step, the plaque grade can be set to high +, making it possible to eliminate the need for the Pg testing step, Tf testing, and Td testing steps.

[0128] Furthermore, while the above embodiments use PCR and fluorescence detection to test for the presence or absence of target bacteria, other methods can be used as long as they can test for the presence or absence of target bacteria in oral samples. For example, the above embodiments use a PCR method in which the test solution is subjected to a predetermined number of thermal cycles, which involve repeated two-phase temperature conditions of heating and cooling. However, other methods, such as a predetermined number of thermal cycles involving repeated three-phase temperature conditions, can also be used, which sequentially repeat the denaturation of DNA from a double-helical structure to single-stranded DNA (approximately 94°C), annealing of the single-stranded DNA to a primer (approximately 55-60°C), and extension of the primer by a thermostable DNA polymerase enzyme (approximately 72°C).

[0129] Furthermore, the specific means of PCR and fluorescence detection used to test for the presence or absence of target bacteria in each of the above embodiments are not limited to the methods disclosed in this specification, and other PCR methods and other fluorescence detection methods can also be used.

[0130] Furthermore, in each of the above embodiments, the sample (specimen) was diluted to a specific dilution ratio before use, but the present invention also includes other dilution ratios as long as the presence or absence of target bacteria can be determined according to predetermined standards. Furthermore, the dilution method and procedure are not important.

[0131] Furthermore, in each of the above embodiments, the multiple grades (multiple grades according to the degree of periodontal disease risk) for classifying oral samples are displayed as "High+", "High", "Medium+", "Medium", and "Low", but these are used for convenience, and in reality, any grade that classifies oral samples into multiple grades according to the degree of periodontal disease risk can be used without restriction.

[0132] Furthermore, in each of the above embodiments, a method for determining the risk of periodontal disease using oral samples has been described, but the present invention can be similarly applied to any specific target disease in which a specific target bacterium is linked to the cause of onset.

[0133] In other words, the present invention can be said to be a method for determining disease risk, which includes an inspection step of inspecting the presence or absence of multiple types of target bacteria in a sample, and an assessment step of classifying the sample into one of multiple levels corresponding to the degree of target disease risk prepared in advance based on the combination of the presence or absence of the multiple types of target bacteria, and determining the target disease risk.

[0134] Conventional testing methods required accurate measurement of the number of bacteria and calculation of the ratio, but the present invention allows the risk of a target disease to be determined simply by testing for the presence or absence of multiple types of target bacteria, allowing for efficient and simple determination.

[0135] Furthermore, in the present invention, it is also possible to set a graded level of importance for each of the plurality of types of target bacteria in advance, and in the determination step, weighting and evaluation can be performed on the test results for the presence or absence of the target bacteria for which a relatively high level of importance has been set. In the above-described embodiment of the present invention, the importance of Pg bacteria is set relatively higher than that of Tf bacteria and Td bacteria, and the importance of Pg type II bacteria is set even higher.

[0136] This configuration allows for more accurate assessment of disease risk, improving usefulness.

[0137] Furthermore, in the present invention, based on the test results for the presence or absence of the target bacteria for which the importance level is set relatively high, it is also possible to distinguish between cases where the test for the target bacteria for which the importance level is set relatively low is omitted and cases where the test is performed. In the second embodiment described above, when Pg bacteria are detected, the tests for Tf bacteria and Td bacteria are omitted and the periodontal disease risk is determined to be "high." Conversely, it is also possible to set a high importance level for target bacteria for which the disease risk can be determined to be "low" if they are not detected. In this case, when the target bacteria are not detected, the disease risk is determined to be "low" and the test for the target bacteria for which the importance level is set relatively low is omitted.

[0138] By configuring it in this way, the risk of disease can be determined more quickly, thereby further improving its usefulness.

[0139] The present invention as described above only requires that the presence or absence of target bacteria can be tested. For example, when using PCR or the like, samples containing DNA or RNA can be used, not limited to oral samples. Furthermore, the target disease is not limited to periodontal disease. For example, a test method can be used in which cell fragments collected by rubbing human skin with a cotton swab are used as a sample, and the risk of skin-related disease is determined based on the detected bacteria. Furthermore, a test method can be used in which cell fragments derived from animals such as dogs or cats are used as a sample, and the risk of disease in the animal is determined based on the detected bacteria. [Explanation of symbols]

[0140] 1. Pg bacteria testing process 2. Tf bacteria testing process 3. Td bacteria testing process 4. Td bacteria testing process 5. Pg type II testing process 10...Separation device 20...Testing methods 21...Amplifier 22...Detection unit 30…Separation means

Claims

1. A method for determining the risk of periodontal disease, which comprises testing the presence or absence of target bacteria in an oral sample, and classifying the oral sample into one of a plurality of grades according to the degree of periodontal disease risk based on the presence or absence of the target bacteria, A method for determining a risk of periodontal disease, wherein the target bacteria include Pg bacteria (Porphyromonas gingivalis), Tf bacteria (Tannellella forsythia), and Td bacteria (Treponema denticola).

2. 2. The method for determining the risk of periodontal disease according to claim 1, wherein the presence or absence of Pg bacteria is tested, and if Pg bacteria is not detected, the presence or absence of Tf bacteria and Td bacteria is tested.

3. The method for determining the risk of periodontal disease according to claim 1 , wherein the target bacteria further include Pg type II bacteria.

4. Check for the presence of Pg bacteria, If Pg bacteria are detected, test for the presence or absence of Pg type II bacteria; The method for determining the risk of periodontal disease according to claim 3, wherein when Pg bacteria are not detected, the presence or absence of Tf bacteria and Td bacteria is examined.

5. A method for determining periodontal disease risk described in any one of claims 1 to 4, wherein the test results for the presence or absence of the target bacteria in the oral sample are compared with a ranking table that associates the presence or absence of the target bacteria with the multiple levels of ranking, and the oral sample is classified into one of the multiple levels of ranking.

6. A method for determining the risk of periodontal disease according to any one of claims 1 to 4, wherein the oral sample is saliva, dental plaque, tongue coating, oral rinse or gingival crevicular fluid.

7. An oral sample separation device, comprising: A testing means for testing the presence or absence of target bacteria in an oral sample; A rating table associating the presence or absence of the target bacteria with multiple levels of rating according to the degree of periodontal disease risk; An oral sample sorting device comprising: a sorting means for comparing the test results for the presence or absence of the target bacteria by the testing means with the grade table and sorting the oral sample into one of the multiple grade levels.

8. The oral sample separation device of claim 7, wherein the testing means comprises an amplification unit that amplifies the DNA of the target bacteria in the test solution containing the oral sample, and a detection unit that tests for the presence or absence of the amplified DNA.

9. 9. The oral cavity sample separation device according to claim 8, wherein the amplification unit utilizes a PCR method.

10. 9. The oral cavity sample separation device according to claim 8, wherein the detection unit utilizes a method for detecting fluorescence from a fluorescent reagent bound to the amplified DNA.

11. The oral sample separation device according to any one of claims 7 to 10, wherein the target bacteria include Pg bacteria (Porphyromonas gingivalis), Tf bacteria (Tannellella forsythia), and Td bacteria (Treponema denticola).

12. The oral cavity sample sorting device according to claim 11, wherein the rating table has at least the following four levels: (1-0) When Pg bacteria are detected (2-1) When Pg bacteria are not detected and both Tf and Td bacteria are detected (2-2) When Pg bacteria are not detected, and either Tf or Td bacteria are detected but the other is not detected (2-3) When Pg bacteria are not detected and neither Tf nor Td bacteria are detected

13. The oral sample separation device according to claim 11 , wherein the target bacteria further include Pg type II bacteria.

14. The oral cavity sample sorting device according to claim 13, wherein the rating table has at least the following five levels: (1-1) When Pg bacteria are detected and Pg type II is detected (1-2) When Pg bacteria are detected but Pg type II is not detected (2-1) When Pg bacteria are not detected and both Tf and Td bacteria are detected (2-2) When Pg bacteria are not detected, and either Tf or Td bacteria are detected but the other is not detected (2-3) When Pg bacteria are not detected and neither Tf nor Td bacteria are detected

15. 8. The oral cavity sample sorting device according to claim 7, further comprising an output unit that outputs information on the grade of the oral cavity sample sorted by the sorting means to an outside of the device.

16. an inspection step of inspecting the presence or absence of multiple types of target bacteria in a sample; A method for determining disease risk, comprising a determination step of classifying the sample into one of a plurality of grades according to the degree of target disease risk prepared in advance based on the combination of the presence or absence of the plurality of types of target bacteria, and determining the target disease risk.

17. A level of importance is set in advance for each of the plurality of types of target bacteria; 17. The method for determining a disease risk according to claim 16, wherein in the determination step, a test result for the presence or absence of the target bacterium, for which a relatively high importance level is set, is weighted for evaluation.

18. A method for determining disease risk as described in claim 17, wherein in the testing process, based on the test results for the presence or absence of the target bacteria for which the importance is set relatively high, the testing of the target bacteria for which the importance is set relatively low is either omitted or tested.

Images