Method for intraoral examination using information on bacterial group correlated with clinical index
阅读说明：本技术 利用与临床指标具有相关性的细菌群的信息的口腔内检查方法 (Method for intraoral examination using information on bacterial group correlated with clinical index ) 是由 外川直之 原爱 村上伸也 野崎刚德 于 2018-11-02 设计创作，主要内容包括：本发明提供一种判定牙周病状态的口腔内检测方法。本发明的口腔内检测方法包括：测定口腔内试样中存在的口腔内细菌群来源的核酸的信号强度,根据该信号强度的测定值计算出所述细菌群的存在比例,将得到的计算值作为指标来判定牙周病的状态,其中,细菌群的存在比例是随牙周袋的数值的增大而增加的细菌种类的细菌量与随牙周袋的数值的增大而减少的细菌种类的细菌量的相关关系。(The invention provides an oral cavity detection method for determining periodontal disease state. The method for detecting in the oral cavity of the present invention comprises: the signal intensity of an intraoral bacterial population-derived nucleic acid present in an intraoral sample is measured, the proportion of the bacterial population present, which is a correlation between the bacterial count of the bacterial species increasing with an increase in the value of the periodontal pocket and the bacterial count of the bacterial species decreasing with an increase in the value of the periodontal pocket, is calculated from the measured value of the signal intensity, and the state of periodontal disease is determined using the calculated value as an index.)
1. An intraoral examination method, the method comprising: measuring the signal intensity of a nucleic acid derived from an intraoral bacterial population present in an intraoral sample, calculating the proportion of the bacterial population present from the measured value of the signal intensity, and determining the periodontal disease state using the calculated value as an index,
the existence ratio of the bacterial group is a correlation between the bacterial amount of the bacterial species increasing with an increase in the numerical value of the periodontal pocket and the bacterial amount of the bacterial species decreasing with an increase in the numerical value of the periodontal pocket.
2. The method according to claim 1, wherein the state of periodontal disease is determined by comparing the obtained calculated value with a cutoff value for the presence ratio of the bacterial population.
3. The method according to claim 1, wherein the proportion of the bacteria group present is a ratio of the bacterial count of the bacterial species that increases with an increase in the value of the periodontal pocket to the bacterial count of the bacterial species that decreases with an increase in the value of the periodontal pocket.
4. The method according to claim 2, wherein the cutoff value is determined based on a ROC curve obtained by calculating a ratio of the presence of the bacterial population from a measured value of signal intensity of an intraoral bacterial population-derived nucleic acid present in the intraoral sample for reference formulation and plotting the calculated value.
5. The method according to claim 1, wherein the existing ratio of the bacterial population is a correlation between the bacterial amount of the Fusobacterium nucleatum species and the bacterial amount of the bacterial species that decreases as the value of the periodontal pocket increases.
6. The method according to claim 1, wherein the bacteria are present in the following ratios (a) and (b):
(a) a correlation between the bacterial count of the bacterial species (including 1 or more species other than the F.nucleatum species) that increase with an increase in the value of the periodontal pocket and the bacterial count of the bacterial species that decrease with an increase in the value of the periodontal pocket;
(b) correlation between the bacterial count of the F.nucleatum species and the bacterial count of the bacterial species that decreased with the increase in the value of the periodontal pocket.
7. The method according to claim 1, wherein the bacterial species increasing with an increase in the value of periodontal pockets is at least one species selected from the group consisting of Porphyromonas gingivalis, Stanemia forskoensis, Treponema denticola, Campylobacter tenuis, Campylobacter rectus, Campylobacter showachii, Clostridium nucleatum Wenychii, Clostridium nucleatum pleomorphus, Clostridium nucleatum subspecies, Clostridium nucleatum, Clostridium paradenticola, Prevotella intermedia, Streptococcus astrus, Actinomyces agglomerans, Airkshiraia denticulata, Protozoa gingivalis, Porphyromonas pulposus, Eubacterium entanglemen, Eubacterium crypthecium, Treponema intermedius and Boromonas sputigena.
8. The method according to claim 1, wherein the bacterial species decreasing with increasing value of periodontal pocket is a bacterial species selected from Prevotella nigrescens, Campylobacter succinogenes, Carbonisatus, Carboxyphilus luteus, Carboxyphilus sputum, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces saprophora, Veillonella parvula, Actinomyces naeslundii II, Pediobacter perus, Prevotella denticola, Prevotella melanogenesis, Gemini coccus haemolytica, Eubacterium borrelia, Corynebacterium malassella, Porphyromonas gingivalis, Microbacterium morganii, Neisseria flavivis, Prevotella lottericola, Micrococcus micronuclei, Actinomyces kudzus, Veillonella typhimurii, Prevotella pallidum, Prevotella charitis, Pasteurella pasteurella, Porphyromonas liquidis, Peptobacterium rubripes, Leptococcus robertni, Prevotella (A. rava, OT308), Roseburia carinii, Chlorella brevicaulis, Streptococcus salivarius, Haemophilus parainfluenza and Streptococcus parahaemolyticus.
9. The method according to claim 5, wherein the Fusobacterium nucleatum species is at least one species selected from the group consisting of Fusobacterium nucleatum Wedneri subsp.
Technical Field
The present invention relates to an intraoral inspection method and the like for determining periodontal disease state by using information on a bacterial population correlated with a clinical index.
Background
Periodontal disease has one side in which it is a bacterial infection associated with various bacteria and one side in which it is a multifactorial disease that progresses in association with pathogenic bacteria (bacterial factors), immunity (host factors) and lifestyle habits, and its onset is associated with periodontal pathogenic bacteria.
As the periodontal pathogenic bacteria, porphyromonas gingivalis, forsteria, treponema denticola, campylobacter rectus, fusobacterium nucleatum, prevotella intermedia, actinomycetemcomitans, etc. have been reported (
Further, it has been reported that actinomycetemcomitans aggregatibacter is a pathogenic bacterium of aggressive periodontitis, and prevotella intermedia is a pathogenic bacterium of adolescent or gestational periodontitis (
As the existing bacterial test for periodontal disease, it has been reported that the total value of 3 kinds of bacteria, "red complex" in plaque or saliva is correlated with the state (degree of progression) of periodontal disease. Specifically, the total number of bacteria of at least 1 of porphyromonas gingivalis, bacteroides forrestii and treponema denticola is determined to be "low" when the total number of bacteria is less than 0.5%, the total number of bacteria is 0.5% or more and less than 5% when the total number of bacteria is "medium", and the total number of bacteria is 0.5% or more when the total number of bacteria is "high" (non-patent document 3).
As a method for detecting periodontal pathogenic bacteria and counting the number of bacteria, for example, a culture method, a method using real-time PCR, next-generation sequencing, and a DNA microarray are reported. In more detail, it is also reported that: the number of cells of porphyromonas gingivalis and/or Bacteroides forsythus (old name: Bacteroides forsythus) in saliva was individually detected by a real-time PCR method (
In addition, a T-RFLP method is also reported, which comprises: genomic DNA of a bacterial flora is recovered and subjected to restriction enzyme treatment, and the bacterial flora is identified from information of fragmented DNA using similarity of patterns as an index (patent document 5). According to this report, patterns of bacterial flora origin having relevance to dental clinical indices were identified. However, no information on the individual specific bacterial numbers is included, nor is it further explained. The T-RFLP method can easily perform comparative analysis of multiple specimens by expressing the structure of bacterial flora as a peak pattern, but on the other hand, it is difficult to grasp the structure of bacterial flora because each peak is not necessarily derived from 1 type of bacteria (non-patent document 4).
As a method for grasping the structure of a bacterial flora, use of a next-generation sequencer has been proposed in recent years. For example, patent document 9 provides an examination method for detecting a salivary bacterial flora by randomly specifying the base sequence of the 16S ribosomal RNA gene of the bacterial flora in a saliva sample collected from a human subject. In this test method, a method for detecting inflammatory bowel disease by saliva is proposed, and as a result of the UniFrac analysis, healthy and healthy populations and CD populations can be identified, but on the other hand, a significant difference test is performed by repeating the t test on various bacteria, and the process of analysis is questionable.
In addition, as a method for capturing the total bacterial count, that is, as a method for detecting total bacteria, the use of a universal primer in which a region highly conserved among microorganisms is selected has been reported (patent documents 6 to 8). In the case of using a DNA microarray, it has been reported that 20 intraoral bacteria are detected by setting 1 set of universal primers in a PCR step for preparing a sample (non-patent
To date, examples of determining the number of at least 1 bacterium in the "red complex" as an indicator of periodontal disease severity have been reported in large numbers. However, since periodontal disease is a disease caused by a variety of bacteria, only information equivalent to that obtained by measuring the periodontal pocket can be obtained in the measurement of a limited variety of malignant bacteria. In addition, the judgment index of the therapeutic effect of periodontal disease is basically based on clinical information obtained from the experience of dentists, and information on bacterial flora is not confirmed in many cases.
In addition, there is no indication of the progression of periodontal disease in the early stages of periodontal disease. Therefore, many patients are aware that periodontal disease has progressed when they are aware of periodontal disease, and even if treatment is started after the symptoms of periodontal disease are recognized, the treatment is not effective in most cases, and thus an effective prediction method relating to the progression of periodontal disease is also required. Specific examples thereof include the following.
"periodontal support therapy (SPT), which is a continuous professional care after completion of periodontal therapy, is an indispensable therapy that maintains" stable disease "and makes the prognosis of periodontal therapy good. Currently, the judgment criteria for the shift to SPT in the clinical field are periodontal pocket probing depth, BOP, and the like. On the other hand, if there is an index for predicting the progress after SPT transfer in the future, it is very useful for judging the treatment plan, but a clear judgment criterion has not been established at present (non-patent document 9).
In such a case, as an attempt to set a determination criterion, the following method has been studied: a method based on a combination of the ratio of the number of porphyromonas gingivalis to the total number of bacteria in saliva and Alanine Aminotransferase (ALT), and a method of measuring the ratio of porphyromonas gingivalis in saliva and the serum antibody titer against porphyromonas gingivalis in combination at the time of re-evaluation of treatment (non-patent document 10). Further, as a predicted index, examination of bacteria in the oral cavity is expected (non-patent document 11), and for example, Haffajee et al report the correlation between the number of bacteria of actinomycete aggregatibacter actinomycetemcomitans and porphyromonas gingivalis and the risk of occurrence of adhesion loss of 2mm or more (non-patent document 12), but as described above, the species to be examined are limited and have not yet been put into practice as a predicted index.
Disclosure of Invention
Problems to be solved by the invention
As described above, currently, there is not sufficient information for determining a treatment course as an index for predicting periodontal disease progression. Accordingly, an object of the present invention is to provide a method for determining the state of periodontal disease and the therapeutic effect of periodontal disease by detecting and quantifying bacteria in the oral cavity in detail by a simple method.
Means for solving the problems
The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the state of periodontal disease can be determined by determining the presence ratio of specific bacteria (groups) with respect to bacteria present in a sample in the oral cavity, and have completed the present invention. The present inventors have also found that the disease condition of the same periodontal pocket value can be further subdivided and classified by measuring all oral cavity bacteria (including periodontal-related bacteria and resident bacteria) that are predominant in plaque at once and creating a model for determining the deterioration of the disease condition based on the presence ratio of periodontal-related bacteria and resident bacteria.
Further, they have found that the therapeutic effect of periodontal disease, the course of periodontal disease, and the like can be determined by obtaining the presence ratio of specific bacteria (group) present in an oral sample, and have completed the present invention.
Namely, the present invention is as follows.
[1] An intraoral examination method, the method comprising: measuring the signal intensity of a nucleic acid derived from an intraoral bacterial population present in an intraoral sample, calculating the proportion of the bacterial population present from the measured value of the signal intensity, and determining the periodontal disease state using the calculated value as an index,
the existence ratio of the bacterial group is a correlation between the bacterial amount of the bacterial species increasing with an increase in the numerical value of the periodontal pocket and the bacterial amount of the bacterial species decreasing with an increase in the numerical value of the periodontal pocket.
[2] The method according to [1], wherein the periodontal disease state is determined by comparing the calculated value with a cutoff value (cutoff value) of the presence ratio of the bacterial population.
[3] The method according to [1] or [2], wherein the proportion of the bacteria existing is a ratio of the amount of bacteria of the bacterial species increasing with an increase in the value of the periodontal pocket to the amount of bacteria of the bacterial species decreasing with an increase in the value of the periodontal pocket.
[4] The method according to [2] or [3], wherein the cutoff value is determined based on a ROC curve obtained by calculating a ratio of the presence of the bacterial population from a measured value of signal intensity of an intraoral bacterial population-derived nucleic acid present in the intraoral sample for reference planning and plotting the calculated value.
[5] The method according to any one of [1] to [4], wherein the ratio of the existence of the bacterial population is a correlation between the bacterial amount of the Fusobacterium nucleatum species and the bacterial amount of the bacterial species that decreases as the value of the periodontal pocket increases.
[6] The method according to any one of [1] to [5], wherein the bacteria exist in the following proportions (a) and (b).
(a) Correlation between the bacterial count of the bacterial species (including 1 or more species other than F. nucleatum species) increasing with the increase in the periodontal pocket numerical value and the bacterial count of the bacterial species decreasing with the increase in the periodontal pocket numerical value
(b) Correlation between the bacterial count of Fusobacterium nucleatum species and the bacterial count of bacterial species that decrease with increasing periodontal pocket number
[7] The method according to any one of [1] to [6], wherein the bacterial species increasing with an increase in the value of periodontal pocket is selected from Porphyromonas gingivalis (Porphyromonas gingivalis), Staphylonella furetianus (tannorella forsythia), Treponema denticola (Treponema pallidum), Campylobacter tenuis (Campylobacter gracilis), Campylobacter procumbens (Campylobacter recuctus), Campylobacter cress and Campylobacter cress (Campylobacter palmatus), Clostridium nucleatum subsp At least one of Eubacterium nodatum (Eubacterium nodatum), Eubacterium crypticum (Eubacterium saphenum), Treponema pallidum (Treponema media), and Geobacillus sputum-producing (Selenomassypiella).
[8] The method according to any one of [1] to [7], wherein the species of bacteria decreasing with an increase in the value of periodontal pocket is selected from Prevotella melanocortina (Prevotella nigrescens), Campylobacter succinogenes (Campylobacter succinogenes), Cappophylobacter gingivalis (Capnocytophaga gingivalis), Cabrophyllobacter lutescens (Capnocytophaga lutescens), Cabrophyllus carbonarius (Capnocytophaga ochracea), Streptococcus gausonii (Streptococcus gordonii), Streptococcus intermedius (Streptococcus intermedius), Streptococcus mitis (Streptococcus mitis), Streptococcus mitis bv 2(Streptococcus mitis bv 2), Actinomyces (Actinomyces nodulis), Vethrobacter parvulus (Vethrobacter parvulus), Streptococcus mitis (Streptococcus mitis II), Streptococcus mitis (Streptococcus mitis), Streptococcus mitis (Bacillus sp), Streptococcus mitis (Streptococcus mitis), Streptococcus mitis (Bacillus subtilis II), Mycoplanaris (Streptococcus mitis), Mycoplanaris (Mycoplanaris), Mycoplanaris (Mycoplanari, Corynebacterium malassense (Corynebacterium glutamicum), Salmonella peptostreatus (Rothia mucoginosa), Porphyromonas catarrhalis (Porphyromonas carotovora), Microbacterium morganii (Solobacterium moorei), Neisseria lutescens (Neisseria flavescens), Prevotella lorescens (Prevotella loescens), Micrococcus megacephala (Megasphaera micturi), Actinomyces geophila (Actinomyces granditzii), Vibrio nonnalis (Veillonella typica), Prevotella albuginella pallidum (Prevotella pallens), Prevotella vulgaris (Prevotella shihii), Porphyromonas (Porphyromonas pasterii), Streptococcus parvus (Prevotella shigella), Streptococcus parahaemophilus (Streptococcus parahaemophilus, Streptococcus parahaemophilus, Streptococcus parahaemophilus (Streptococcus parahaemophilus).
[9] The method according to any one of [5] to [8], wherein the Fusobacterium nucleatum species is at least one species selected from the group consisting of Fusobacterium nucleatum Wen subspecies, Fusobacterium nucleatum multiforme subspecies, Fusobacterium nucleatum animal subspecies and Fusobacterium nucleatum subspecies.
[10] An intraoral examination method, the method comprising: the signal intensity of an intraoral bacterial flora-derived nucleic acid present in an intraoral sample is measured, the proportion of the bacterial flora present is calculated from the measured value of the signal intensity, and the calculated value is correlated with the state, course or therapeutic effect of periodontal disease.
[11] The method according to [10], wherein the correlation between the proportion of bacteria existing and the state of periodontal disease is performed by calculating a ratio of the bacteria species increasing with an increase in the numerical value of periodontal pocket to the bacteria species decreasing with an increase in the numerical value of periodontal pocket and/or the bacteria species increasing with a decrease in the numerical value of periodontal pocket.
[12] The method according to [10] or [11], wherein the correlation between the existence ratio of the bacterial group and the course of periodontal disease is performed by calculating a ratio of the bacterial species belonging to the genus Clostridium which increase with an increase in the numerical value of periodontal pocket to the bacterial species which decrease with an increase in the numerical value of periodontal pocket and/or the bacterial species which increase with a decrease in the numerical value of periodontal pocket.
[13] The method according to any one of [10] to [12], wherein the correlation between the proportion of bacteria existing and the therapeutic effect of periodontal disease is performed by comparing the following values of (a) and/or (b) before and after the treatment of periodontal disease.
(a) The ratio of the number of bacteria increasing with the increase of the number of periodontal pockets to the number of bacteria decreasing with the increase of the number of periodontal pockets and/or the number of bacteria increasing with the decrease of the number of periodontal pockets
(b) The ratio of the bacterial species belonging to the genus Clostridium which increase with the increase in the number of periodontal pockets to the bacterial species which decrease with the increase in the number of periodontal pockets and/or the bacterial species which increase with the decrease in the number of periodontal pockets.
[14] The method according to [12] or [13], wherein the bacterial species belonging to the genus Clostridium is at least one species selected from the group consisting of Fusobacterium nucleatum Wedneri subsp, Fusobacterium nucleatum multiforme subsp, Fusobacterium nucleatum animal subsp, Fusobacterium nucleatum subsp and Fusobacterium periodontopanum.
[15] The method according to any one of [11] to [14], wherein the bacterial species increasing with an increase in the value of the periodontal pocket is at least one species selected from the group consisting of Porphyromonas gingivalis, Fostana veneris, Treponema denticola, Campylobacter tenuis, Campylobacter rectus, Showa Campylobacter showa, Clostridium nucleatum Wenitzschia, Clostridium nucleatum pleomorphus, Clostridium nucleatum subspecies, Clostridium nucleatum, Clostridium paradenticola, Prevotella intermedia, Streptococcus astrus, Actinomyces and Airkia rodensis,
the species of bacteria that decrease with an increase in the numerical value of the periodontal pocket and/or the species of bacteria that increase with a decrease in the numerical value of the periodontal pocket are at least one selected from Prevotella nigrescens, Campylobacter succinogenes, Carbonocytophaga gingivalis, Carbonocytophaga fulvescens, Carbonocytophaga sputigena, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces carinii, Veillonella parvula, Actinomyces naeslundii II, and harmful Porphyromonas lunata.
[16] The method according to any one of [12] to [15], wherein the bacterial species belonging to the genus Clostridium is at least one species selected from the group consisting of Fusobacterium nucleatum Wedneri subsp, Fusobacterium nucleatum multiforme subsp, Fusobacterium nucleatum animal subsp, Fusobacterium nucleatum subsp and Fusobacterium periodontopanum,
the species of bacteria that decrease with an increase in the numerical value of the periodontal pocket and/or the species of bacteria that increase with a decrease in the numerical value of the periodontal pocket are at least one selected from Prevotella nigrescens, Campylobacter succinogenes, Carbonocytophaga gingivalis, Carbonocytophaga fulvescens, Carbonocytophaga sputigena, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces carinii, Veillonella parvula, Actinomyces naeslundii II, and harmful Porphyromonas lunata.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, a large number of oral bacteria (including periodontal-related bacteria and resident bacteria) can be detected and quantified at a time, and the state of periodontal disease can be determined more finely than in the conventional method. In addition, according to the present invention, the state of periodontal disease, the therapeutic effect of periodontal disease, and the course of periodontal disease can be determined, and the disease state with the same periodontal pocket size can be classified into more subdivided states. In addition, the treatment effect and stable state of the disease can be determined even if the periodontal pocket value is not present. Further, the judgment model performance can be improved by replacing the bacterial species used in the judgment in the future.
Drawings
FIG. 1-1 is DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 types of bacteria mounted on the DNA chip are shown in the figure.
FIG. 1-2 is DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIGS. 1 to 3 are DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIGS. 1 to 4 are DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIGS. 1 to 5 are DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIGS. 1 to 6 are DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIGS. 1 to 7 are DNA chip measurement data (SN ratio) of subgingival plaque collected from 220 male and female subjects aged 20 to 70 before periodontal disease treatment: longitudinal axis and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The 28 kinds of bacteria mounted on the DNA chip are shown in the figure.
FIG. 2 is a balance index of bacterial flora ("15 positive-related flora" and 13 negative-related flora "along the longitudinal axis) and depth of periodontal pocket (Pd): scatter plot of the horizontal axis. The figure shows 220 names of data.
FIG. 3 shows the balance index (LOG10 transformation) of the bacterial population of the periodontal pocket 1-3 mm (defined as non-disease group) and the periodontal pocket 5mm or more (defined as disease group): graph of histogram of vertical axis (frequency: horizontal axis).
Fig. 4 is a graph showing the result of ROC analysis of the balance index.
FIG. 5 is a radar chart showing SN ratios of respective bacteria in samples having a periodontal pocket value of 4mm and a balance index (LOG10) value of 1.516648. The axis from the center toward the outside is a balance index.
FIG. 6 shows the balance index of the flora ("disease progression index bacteria" 2 species, "negative relative bacteria" 13 species): longitudinal axis and periodontal pocket probing depth (Pd): scatter plot of the horizontal axis. The figure shows 220 names of data.
FIG. 7 shows the balance index (LOG10 transformation) of the bacterial population at 1-3 mm periodontal pocket (defined as non-disease group) and at 5mm periodontal pocket or more (defined as disease group): graph of histogram of vertical axis (frequency: horizontal axis).
Fig. 8 is a graph showing the result of ROC analysis of the balance index.
FIG. 9 is a radar chart showing SN ratios of respective bacteria in samples having a periodontal pocket value of 4mm and a balance index (LOG10) value of 0.883. The axis from the center toward the outside is a balance index.
Fig. 10 is a diagram showing the results of the determination and subdivision of the periodontal disease state.
FIG. 11-1 is a radar chart showing the SN ratio of each bacterium in a sample in a state after being fragmented. The axis from the center toward the outside is a balance index.
FIG. 11-2 is a radar chart showing the SN ratio of each bacterium in a sample in a state after being fragmented. The axis from the center toward the outside is a balance index.
FIG. 11-3 is a radar chart showing the SN ratio of each bacterium in a sample in a state after being fragmented. The axis from the center toward the outside is a balance index.
Fig. 12 is a graph showing the result of ROC analysis of the balance index.
Fig. 13 is a graph showing the result of ROC analysis of the balance index.
Fig. 14 is a scattergram of the balance index, and depth of periodontal pocket before and after treatment.
Fig. 15 is a scattergram (prepared from copy number) of the balance index, and depth of periodontal pocket before and after treatment.
Fig. 16 is a graph showing the relative ratio of each bacteria derived from a sample obtained by the analysis result of the next-generation sequencer.
FIG. 17 is a scatter plot of balance index and periodontal pocket depth (Pd).
FIG. 18 is a histogram of data regarding the periodontal pocket depths of 1 to 3mm and 5mm or more among the data shown in FIG. 17.
Fig. 19 is a graph showing the results of performing ROC analysis.
Detailed Description
The present invention will be described in detail below. The scope of the present invention is not limited to the above description, and can be modified and implemented as appropriate without departing from the spirit of the present invention, in addition to the following examples. All publications cited in the present specification, such as prior art documents and publications, patent publications, and other patent documents, are incorporated herein by reference.
The present invention is an intraoral examination method including: the signal intensity of a nucleic acid derived from an intraoral bacterial flora present in an intraoral sample is measured, the proportion of the bacterial flora present is calculated from the measured value of the signal intensity, and the periodontal disease state is determined using the calculated value as an index. In the present invention, the existence ratio of the bacterial flora refers to a correlation between the bacterial count of the bacterial species increasing with an increase in the numerical value of the periodontal pocket and the bacterial count of the bacterial species decreasing with an increase in the numerical value of the periodontal pocket.
The specific embodiment of the method for examination in the oral cavity is not limited, and the method for measuring the number of bacteria in an oral cavity sample will be described mainly by using a DNA chip.
The detection, measurement, and quantification of bacteria in the oral sample may also be performed by methods other than the method using a DNA chip, for example, an invader method, a real-time PCR method, an invader PCR method, a next-generation sequencing method, and the like.
1. Oligonucleotide probe for detecting bacteria in oral cavity
In the method of the present invention, when detecting oral bacteria from an oral sample collected from a subject, a DNA chip on which at least one of the following probes (b) and (c) and probe (a) can be mounted can be used.
(a) Probes comprising nucleic acids that specifically hybridize to genes (or gene-derived amplification products) of bacteria to be detected
(b) Total amount index probe composed of nucleic acid hybridizing with gene (or gene-derived amplification product) of all bacteria
(c) Probes consisting of nucleic acids which hybridize specifically with 1 or more absolute quantitative indices, respectively
In general, a DNA chip is a generic term referring to a substrate on which probes are arranged. In the present specification, the names of DNA chips, DNA microarrays and the like are treated as synonyms without distinction.
(1) Oral bacteria to be measured
In the detection method of the present invention, the oral bacteria to be measured are not limited, but may be those belonging to the genus Porphyromonas, Tenera, Treponema, Prevotella, Campylobacter, Clostridium, Streptococcus, Acreuterium, Carborunophilus, Eikezia, Actinomyces, Veillonella, Oenomonas, Pseudomonas (Pseudomonas), Haemophilus (Haemophilus), Klebsiella (Klebsiella), Serratia (Serratia), Moraxella (Moraxella), Eubacterium (Eubacterium), Microbacterium (Parvimonas), Produce (Filifoctor), Prevotella (Alloprovella), Microbacterium (Solobacterium), Rosteia (Rothia), Peptostreptococcus (Peptococcus), Genetococcus (Geebella), short-chain bacterium (Corynebacterium), Gracillus (Gracillus), Megasphaera (Megasphaera) and SR1 phylum bacteria as detection target strains.
More specifically, for example, Porphyromonas gingivalis, Fusobacterium venenatum, Treponema denticola, Campylobacter tenuis, Campylobacter rectus, Campylobacter showayae, Clostridium nucleatum Wedneri subsp, Clostridium nucleatum Bacteroides, Clostridium nucleatum subsp, Clostridium nucleatum, Clostridium paradenticola, Prevotella intermedia, Prevotella nigrescens, Streptococcus astrus, Actinomyces actinomycetemcomitans, Campylobacter succinogenes, Carboruncatus, Carbonocardia lutescens, Carborundum carotovorans, Endochromobacter cerealis, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces saprophora, Veillonella parvula, Actinomyces naeus II, harmful Streptococcus lunatus, Streptococcus sanguis (Streptococcus sanguis sanguinis) and Streptococcus mutans, Actinomyces viscosus (Actinomyces viscosus), Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus pneumoniae (S streptococcuspneumoniae), Streptococcus mutans, Eubacterium tangles, Micromonospora parvum, Progingival cremastra gingivalis, Streptococcus sobrinus, Porphyromonas pasteurii, Veillonella sarmentosa, Haemophilus parainfluenzae, Prevotella (A. rava, OT308), Streptococcus paracasei, Actinomyces chlamydiae, Prevotella pallidus, Prevotella lorella rosenbergii, Prevotella histidinella, Microbacterium morganii, Prevotella melanogenes, Bordetella expectorans, Rosemophilus carinii, Rosemophilus peptis, Legobacter rosei, Streptococcus gastri, Prevotella denticulatus, Porphyromonas pulposus, Streptococcus salivarius, Micrococcus intermedius, Actinomyces, Streptococcus hemolyticus, Spirosoma, Porphyromyces, Porphyromonas pini, and Malloti, Bacteria such as Eubacterium crypthecogenicum, Neisseria flavivis, Pediococcus brevis, Eubacterium interjalis, Megasphaera nucleatum, Prevotella charantia, and SR1 sp.OT 345 are detection target bacteria species, and more preferably, the increase or decrease of the bacterial species is clear in relation to the disease state.
In order to detect the state in the oral cavity, for example, a species which clearly increases or decreases along with the value of the periodontal pocket may be cited.
In the present invention, the "value of periodontal pocket" refers to the value of the depth (Pd) of the periodontal pocket. The depth (Pd) of the periodontal pocket is the distance from the gingival margin to the tip of the periodontal probe when the periodontal probe is inserted into the pocket. The numerical values were expressed in units of 1 mm. Here, the "periodontal probe" refers to a pouch measurement instrument (period-probe).
The increase or decrease is not limited to a pattern of increase or decrease such as a bacterial population in which an increase is observed when the value of the periodontal pocket is large, or a bacterial population in which the bacterial count is retained when the increase is started when the value of the periodontal pocket is small and then the increase is continued when the value of the periodontal pocket is large. As simpler examples, there are a group of "bacterial species increasing with an increase in the number of periodontal pockets" and a group of "bacterial species decreasing with an increase in the number of periodontal pockets".
The bacterial species that increase with an increase in the numerical value of the periodontal pocket and the bacterial species that decrease with an increase in the numerical value of the periodontal pocket can be confirmed by a tool that can measure the bacterial count (or a measured count proportional to the bacterial count such as the SN ratio). The tool is not particularly limited, and for example, a DNA chip can be used.
When confirming using the DNA chip, the intraoral sample is measured using the DNA chip, and then the correlation coefficient between the value of the periodontal pocket and the measured amount such as the bacterial count or SN ratio of each bacterium is calculated, and a bacterial group having a positive correlation coefficient and a bacterial group having a negative correlation coefficient can be classified and identified. In the case where the number of measurements is 40 or more, the absolute value of the correlation coefficient is preferably 0.02 or more, more preferably 0.1 or more, still more preferably 0.2 or more, particularly preferably 0.4 or more, and most preferably 0.6 or more.
When data after experimental error correction is used for determination of periodontal disease state, data after experimental error correction is also used for classification of bacterial groups.
The types of bacteria that increase with an increase in the number of periodontal pockets (hereinafter, sometimes referred to as "positively-related bacteria") are bacteria that increase with the progression of periodontal disease. Porphyromonas gingivalis, Forstanemia furiosaensis, Treponema denticola and the like are known and used for conventional examination of periodontal disease bacteria.
Examples of the bacterial species increasing with the increase in the number of periodontal pockets include at least one species selected from the group consisting of Porphyromonas gingivalis, Statina fossilia, Treponema denticulata, Campylobacter tenuis, Campylobacter rectus, Campylobacter showachii, Clostridium nucleatum Wenyi, Clostridium nucleatum pleomorphus, Clostridium nucleatum subspecies, Clostridium nucleatum, Clostridium paradenticola, Prevotella intermedia, Streptococcus astrus, Actinomyces agglomerans, Exkernella rodensis, Vibrio gingivalis, Porphyromonas pulposus, Eubacterium entanglemen, Eubacterium crypthecium, Treponema intermedius, and Porphyromonas lunata.
Among them, bacteria that start increasing when the value of the periodontal pocket is small and then increase and retain the bacterial amount when the value of the periodontal pocket becomes large are sometimes referred to as "disease progression index bacteria" hereinafter. The "disease progression index bacteria" are considered to play a role of connecting "harmful bacteria" and "beneficial bacteria" described later, and to serve as an index of an early stage of the progression of periodontal disease. Specific examples of the "disease-progression index bacteria" include Fusobacterium nucleatum strains.
Examples of the Fusobacterium nucleatum species include at least 1 species selected from the group consisting of Fusobacterium nucleatum Wedneri, Fusobacterium nucleatum Bacteroides, Fusobacterium nucleatum animal subspecies, and Fusobacterium nucleatum subspecies.
On the other hand, the bacteria group that is found to increase when the value of the periodontal pocket is increased may be hereinafter referred to as "harmful bacteria". Specific examples of the "harmful bacteria" include bacteria other than the Fusobacterium nucleatum species among the bacteria species that increase with the increase in the number of periodontal pockets. Examples thereof include at least 1 selected from the group consisting of Porphyromonas gingivalis, Stannia forskoensis, Treponema denticola, Campylobacter tenuis, Campylobacter rectus, Campylobacter showae, Clostridium paradenticola, Prevotella intermedia, Streptococcus astrus, Actinomyces reuteris, Exkenella denticulata, Protozoa gingivalis, Porphyromonas pulposus, Eubacterium entanglemenus, Eubacterium crypthecium, Treponema intermedius and Boromonas sputigena.
Examples of the types of bacteria that decrease with an increase in the value of periodontal pockets (hereinafter, sometimes referred to as "negative-related bacteria") include some of bacteria belonging to the genus Streptococcus, Actinomyces, Vellonella, and the like.
These species include: (i) bacteria that decrease with an increase in the number of periodontal pockets (i.e., deterioration of periodontal disease), (ii) bacteria species that increase with a decrease in the number of periodontal pockets (improvement of periodontal disease), or both (i) and (ii) above. Hereinafter, the bacterial population that decreases with an increase in the number of periodontal pockets may be referred to as "beneficial bacteria".
Examples of the species of bacteria that decrease with an increase in the value of periodontal pocket and/or that increase with a decrease in the value of periodontal pocket include those selected from Prevotella nigrescens, Campylobacter succinogenes, capnocytophaga gingivalis, capnocytophaga lutescens, capnocytophaga sputigena, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces carinii, MicroWeironella parvula, Actinomyces naeslundii II, Pediomonas perus, Prevotella denticola, Prevotella melanogenes, Streptococcus hemolyticus, Eubacterium borgpoensis, Corynebacterium malassella marxianus, Porphyromonas casinosa, Microbacterium morganii, Neisseria flaviviridae, Prevotella loensis, Micrococcus putida, Actinomyces parvum, Actinomyces kudzus, atypical Veillonella virescens, Prevotella pallidus, Proteus, Microbacterium pallidum, and Microbacterium crepidae, At least one of Prevotella charantia, Porphyromonas pasteurii, Rogomphrena rosenbergii, Prevotella (A.rava, OT308), Rostella carinii, Pediococcus brevicaulis, Streptococcus salivarius, Haemophilus parainfluenza and Streptococcus parahaemolyticus.
(2) Concerning the probe (a)
In the present invention, the oligo-DNA used as the probe (a) is an oligo-DNA which can hybridize with a base sequence of a bacteria-specific region (a region whose base sequence varies depending on the type of bacteria) in a base sequence of a nucleic acid derived from bacteria in the oral cavity. Here, the nucleic acid is not limited as long as it is any of DNA and RNA including chromosomal DNA, plasmid DNA, and the like, and chromosomal DNA is preferable. Specifically, the oligonucleotide used as a probe in the present invention is an oligonucleotide capable of hybridizing with the base sequence of the 16S rRNA gene in the chromosomal DNA of the above-mentioned oral bacteria.
The probe usable in the present invention is preferably designed by selecting a region having a nucleotide sequence specific to each of the above-mentioned oral bacteria to be detected and designing the nucleotide sequence of the region. In general, when designing a probe, it is necessary to select a specific region, and to have a uniform melting temperature (Tm) and to make it difficult to form a secondary structure.
For example, a base sequence specific to each of the oral bacteria can be found by designing a probe in a region different between species by a multiple sequence alignment or the like. The algorithm for performing the alignment is not particularly limited, and as a more specific analysis program, for example, a program such as clustalx1.8 can be used. The parameters for the alignment may be executed in a default state of each program, and may be appropriately adjusted according to the type of the program.
On the other hand, the specificity of the probe may be such that bacteria of the same genus are all detected at once based on the specificity at the genus level, or may be such that detection can be performed at the level of each species, and may be appropriately determined depending on the purpose of bacteria detection. Depending on the level of specificity of detection, the detectable bacterial species may be limited to a particular 1 species, or may be the sum of the genus levels (total).
(3) Concerning the probe (b)
The total amount indicator probe is a probe that can be amplified with a specific primer set and is used to capture all bacteria in a sample. In detecting bacteria, it is also extremely important to detect the total amount of bacteria from the viewpoint of the ratio of the degree of bacteria to be detected to the total amount of bacteria including bacteria not to be detected, and the amount of bacteria present in the original sample. Bacteria that are not the object of detection are understood to be the sum (total) of bacteria whose presence and type are known but not the object of detection and bacteria whose presence and type are unknown.
In order to detect the total amount of bacteria, for example, the total amount of bacteria may be measured separately from the DNA chip, but the DNA chip may be loaded with a probe as an index of the total amount of bacteria, thereby improving the ease of operation. As the probe, a base sequence common to a plurality of bacterial species can be used as the base sequence amplified by the primer set. When such a sequence is not found, a plurality of relatively common sequences can be designed and comprehensively determined to be used as a total amount indicator probe. The total amount indicator probe is preferably a probe that hybridizes to a nucleic acid derived from a bacterium contained in a sample, and more specifically, a probe that hybridizes to a base sequence that is common to a plurality of types of bacteria to be detected among the base sequences amplified by the specific primer pair.
The total amount index indicates the total amount of the amplified product specific to each species, and therefore the amount is usually large, and thus the target signal intensity sometimes exceeds the range of detectable signal intensity. In order to prevent such a situation, it is desirable to limit the amount of the detection substance to be used for hybridization. Alternatively, when designing a probe, for example, the Tm value of the probe is lowered. Specifically, a method of reducing the GC content and shortening the sequence length of the probe itself can be considered.
In addition, when hybridization is performed, a decrease in signal intensity can be achieved by adding a nucleic acid that has a competitive effect on hybridization of the amplified nucleic acid with the total amount index probe. Examples of such nucleic acids include nucleic acids having a sequence identical to all or part of the total amount indicator probe, or nucleic acids having a sequence complementary to all or part of the total amount indicator probe.
(4) About the probe (c)
The absolute amount index probe is a probe that hybridizes to only the nucleic acid of the absolute amount index. In the present specification, the absolute amount index is an index indicating the amount of nucleic acid added to a sample in a predetermined amount before an amplification reaction or a hybridization reaction. The absolute amount index is a nucleic acid that can reliably undergo an amplification reaction as long as a normal amplification reaction is performed, and functions as a so-called positive control. Therefore, if a probe specific to the absolute amount index is mounted on the DNA chip in advance, whether or not the amplification reaction, hybridization, or the like has been properly performed can be confirmed from the detection result.
If the absolute amount indicator is added before the amplification reaction, a specific primer set for the absolute amount indicator needs to be added to the reaction solution in advance, and the absolute amount indicator may be amplified commonly by a primer set for bacteria as the case may be. In addition, in order to detect the target bacteria independently of other detection targets by hybridization, it is necessary to select a base sequence having low similarity to both the detection target bacteria and the non-detection target bacteria.
When the amplification efficiency and the hybridization efficiency slightly increase or decrease when the absolute amount index is set to 1 type, the correction coefficient may be calculated by comparing the signal intensities of the absolute amount indexes. When data of a plurality of DNA chips are compared, the comparison can be performed based on the signal intensity corrected by the correction factor.
Specific examples of the probes (a), (b), and (c) are shown in Table 1.
Examples of the specific probes for each bacterium are shown in table 1. (SEQ ID NO: 1 to 33).
Sequence number 34 shows an example of a total amount index probe.
The sequence number 35 shows an example of an absolute quantity index probe.
The serial number 36 shows an example of an absolute quantity index.
TABLE 1
Serial number
< design of primer >
In the primer design method of the present invention, first, at least one variable region showing the diversity of the bacteria to be analyzed is selected, and a universal primer design region having high conservation is selected before and after the selected variable region to design a primer sequence. The variable region to be used is not limited, and the 16S rRNA gene and the like of all bacteria can be exemplified in the genome sequence. In the 16S rRNA gene, it is preferable to target at least one region of the full-length or variable regions V1-V9. More preferably, the variable regions V1-V6 are targeted. It is further preferable that the variable domains V3-V6 be targeted. It is noted that the variable region of the 16S rRNA gene is known to consist of the region V1-V9, which has been identified.
In order to evaluate the comprehensiveness of primers, a database in which genomic sequences of bacteria are obtained in a wide range is utilized. Specific examples thereof include RDP, NCBI, KEGG, and MGDB. As an example, the designed universal primer sequence is entered into the ProbeMatch of the database of RDP. The number of perfect matches in the total search can be obtained in the list of results. The closer the number of perfect matches is to the total search, the higher its comprehensiveness. At this time, string may select "Type" as a condition. In addition, Sourse may select "Isolates".
When designing a sequence for an absolute value index and a primer sequence for amplification thereof, X integers of 1 to 4 (X is an arbitrary number) are randomly generated by using the RNDB19ETWEEN function of software "EXCEL" (manufactured by MICROSOFT Co., Ltd.), and are connected to form a numerical value of X digit consisting of only numerical values of 1 to 4, and the numerical values of 1 to 4 are substituted by any letters of A, G, C and T, thereby obtaining a random sequence. For example, a large number of random sequences based on X-base ATGC can be obtained by substituting 1 with A, 2 with T, 3 with C, and 4 with G.
Of these sequences, only those sequences having the same sum of G and T as that of A and T were selected, and the selected sequences were subjected to Blast search using a database such as GenBank of NCBI, and nucleic acids having a small number of similar sequences were selected for nucleic acids derived from organisms.
In order to keep the reaction efficiency at the time of amplification reaction as constant as possible, it is desirable that the length of the amplified base in the bacteria to be detected is not greatly different from the length of the amplified base in the absolute amount index. For example, if the amplification product of the bacteria to be detected is about 500bp, it is desirable that the amplification product of the absolute amount index is about 300bp to 1000 bp.
On the other hand, in the case where the length of the amplified strand is confirmed by electrophoresis or the like after amplification, it is designed so that the amplified strand has a length different from that of the bacteria to be detected, and then the amplified product derived from the absolute amount indicator is detected at a position different from the band of the bacteria to be detected, and the success or failure of the amplification reaction is confirmed before hybridization.
Finally, when the concentration of the absolute amount indicator contained in the sample is too high, the competition with the bacteria to be detected in the amplification reaction becomes severe, and the bacteria to be detected which should be originally detected may become undetectable, and therefore, it is necessary to appropriately adjust the concentration according to the application.
When the nucleic acid derived from bacteria and the absolute amount indicator are amplified separately, a multiplex method using 2 or more primer pairs can be applied as necessary. On the other hand, a method of competing the primers in a common set may be used as needed.
Examples of primer sequences are shown in Table 2. A pair of primers for bacterial amplification (SEQ ID NOS: 37 and 38) and a pair of primers for absolute quantity indicator (SEQ ID NOS: 39 and 40) can be used. In addition, the primers shown in Table 3 can also be used.
TABLE 2
Serial number
Remarks for note
37
Forward primer (for bacterial amplification)
Fluorescence labeling at 5
TCCTACGGGAGGCAGCAGT
38
Reverse primer (for bacterial amplification)
CAGGGTATCTAATCCTGTTTGCTACC
39
Forward primer (for absolute quantity index amplification)
Fluorescence labeling at 5
GAGAAGCCTACACAAACGTAACGTC
40
Reverse primer (for absolute quantity index amplification)
CTCTAAGACCGCTCTATCTCGG
TABLE 3
Serial number
Primer name
Remarks for note
Sequence of
41
Cy5-Universal16S-FWD
Fluorescence labeling at 5
TCCTACGGGAGGCAGCAGT
42
Cy5-Universal16S-FWD1
Fluorescence labeling at 5
TCCTACGGGAGGCAGCAG
43
Cy5-Universal16S-FWD2
Fluorescence labeling at 5
TCCTACGGGAGGCAGCA
44
Cy5-Universal16S-FWD3
Fluorescence labeling at 5
TCCTACGGGAGGCAGC
45
Cy5-Universal16S-FWD4
Fluorescence labeling at 5
CCTACGGGAGGCAGC
46
Cy5-Universal16S-FWD5
Fluorescence labeling at 5
CTACGGGAGGCAGCAG
47
Cy5-Universal16S-FWD6
Fluorescence labeling at 5
TACGGGAGGCAGCAG
48
SidneyU RVS
GGACTACCAGGGTATCTAATCCTGTT
49
Universa RVS2 2014
CAGGGTATCTAATCCTGTTTGCTACC
50
Universal RVS1 2016
CAGGGTATCTATCCTGTTYG
51
Universal RVS2 2016
CAGGGTATGTATCCTGTT
52
Universa RVS3 2016
GGGTATCTATCGYGTT
53
Universal RVS4 2016
CRGGGTATCTATCCYGTT
< design of Probe >
When designing the probe used in the present invention, the length of the probe is not limited, but is, for example, preferably 10 bases or more, more preferably 16 to 50 bases, and still more preferably 18 to 35 bases. If the length of the probe is appropriate (if the length is in the above range), nonspecific hybridization (mismatch) can be suppressed, and the probe can be used for specific detection. In designing the probe used in the present invention, Tm is preferably confirmed in advance. The Tm is a temperature at which 50% of an arbitrary nucleic acid strand forms a hybrid with its complementary strand, and the hybridization temperature needs to be optimized in order to form a double strand between a template DNA or RNA and a probe and perform hybridization. On the other hand, if the temperature is lowered, nonspecific reactions are likely to occur, so that it is desirable that the temperature be as high as possible.
Therefore, the Tm of the nucleic acid fragment to be designed is an important factor in hybridization. As the software that can be used in the present invention, for example, Probe Quest (registered trademark; DYNACOM Co., Ltd.) and the like can be cited. Further, the Tm can be confirmed by self-calculation without using software. In this case, a calculation formula based on a Nearest Neighbor Method (Nearest Neighbor Method), wallence Method, GC% Method, or the like can be used. The probe of the present invention is not limited, and the average Tm is preferably about 35 to 70 ℃ or 45 to 60 ℃. As conditions for specific hybridization as a probe, conditions for GC content and the like are known to those skilled in the art.
The probe of the present invention may further comprise an additional sequence such as a tag sequence. The tag sequence may be a spacer sequence such as "AAAAAAA". In the method of the present invention, the base sequence of the nucleic acid of the oral bacteria to be detected is not necessarily the base sequence itself in all cases, and may be a base sequence in which a part of the base sequence is mutated by deletion, substitution, insertion, or the like. Therefore, the base sequence of the nucleic acid to be detected can be a mutant gene which hybridizes to a sequence complementary to the base sequence under stringent conditions and has a function and activity derived from each base sequence, and the probe can be designed based on the base sequence of the mutant gene.
Specifically, the designed probe includes the sequence of the probe (a). Further, it is preferable to use a substance containing a DNA having a function of hybridizing with a DNA consisting of a base sequence complementary to these DNAs under stringent conditions and capable of detecting at least a part of the base sequence of a nucleic acid derived from an oral bacterium. The nucleotide sequence of such a DNA is preferably a nucleotide sequence having at least 60% identity to the probe (a), more preferably 80% or more, further preferably 90% or more, further preferably 95% or more, and particularly preferably 97% or more.
In practice, when the probe is used for detection, the stringency of hybridization must be taken into consideration. By setting the stringency to a certain degree, even if there are regions of similar base sequences among specific regions in the respective nucleic acids of various intraoral bacteria, hybridization can be performed with distinction from other different regions. In addition, when the base sequences of the specific regions are substantially different from each other, the stringency can be set moderately.
As such stringent conditions, for example, stringent conditions are hybridization at 50 to 60 ℃ and mild conditions are hybridization at 30 to 40 ℃. Among the conditions for hybridization, stringent conditions include, for example, "0.24M Tris. HCl/0.24M NaCl/0.05% Tween-20," 40 ℃ "," 0.24M Tris. HCl/0.24M NaCl/0.05% Tween-20, "37 ℃", "0.24M Tris. HCl/0.24M NaCl/0.05% Tween-20,", and 30 ℃ ", and more stringent conditions include, for example," 0.24M Tris. HCl/0.24M NaCl/0.05% Tween-20, "50 ℃", "0.24M Tris. HCl/0.24M NaCl/0.05% Tween-20," 55 ℃ "," 0.06M. Tris. HCl/0.06M NaCl/0.05% Tween-20, ", and" 60 ℃ ".
More specifically, there is a method of: the probe was added and the mixture was kept at 50 ℃ for 1 hour or more to form a hybrid, and then washed 4 times at 50 ℃ for 20 minutes in 0.24M Tris & HCl/0.24M NaCl/0.05% Tween-20, and finally washed 1 time at 50 ℃ for 10 minutes in 0.24M Tris & HCl/0.24M NaCl. By raising the temperature at the time of hybridization or washing, more stringent conditions can be set. As a person skilled in the art, conditions other than the conditions such as the salt concentration of the buffer and the temperature may be set in consideration of various conditions such as the probe concentration, the length of the probe, and the reaction time. For detailed procedures of the hybridization method, reference may be made to "Molecular Cloning, A Laboratory Manual 4 the." (Cold Spring Harbor Press (2012), "Current Protocols in Molecular Biology" (John Wiley & Sons (1987-1997)) and the like.
The nucleotide constituting the probe used in the present invention may be either DNA or RNA or PNA, or a hybrid of 2 or more kinds of DNA, RNA and PNA. For example, it can be prepared by chemical synthesis (purification by HPLC or the like) using a usual oligonucleotide synthesis method. Further, a nucleotide obtained by chemically modifying the end or the middle of the above-mentioned nucleotide may be used.
2. DNA chip for detecting gene of bacteria in oral cavity for use in measurement of amount of bacteria in oral cavity
As described above, in the method of the present invention, a DNA chip in which a plurality of the oligonucleotide probes described in the
Another preferable form of the support is a form using hollow fibers. In the case of using the hollow fiber as a support, preferable examples can be given: a DNA chip (hereinafter, also referred to as "fiber-type DNA chip") is obtained by immobilizing a given probe for each type on each hollow fiber, bundling and immobilizing all the hollow fibers, and then repeatedly cleaving the fibers in the longitudinal direction. The microarray can also be described as a type in which nucleic acids are immobilized on a through-hole substrate, also called a so-called "through-hole type DNA chip" (refer to Japanese patent No. 3510882, etc.).
The method for immobilizing the probe on the support is not limited, and any binding method may be used. Further, the probe is not limited to the case of being directly immobilized on the support, and for example, the support may be coated with a polymer such as polylysine in advance, and the probe may be immobilized on the treated support. When a tubular body such as a hollow fiber is used as the support, a gel may be held in the tubular body, and the probe may be fixed to the gel. Hereinafter, a fiber-type DNA chip, which is one embodiment of the DNA chip, will be described in detail. The DNA chip can be produced, for example, by performing the following steps (i) to (iv).
(i) A step of three-dimensionally arranging a plurality of hollow fibers so that the longitudinal directions of the hollow fibers are in the same direction to produce an arrangement body
(ii) Embedding the array to produce a block
(iii) Introducing a gel precursor polymerizable solution containing an oligonucleotide probe into the hollow portion of each hollow fiber of the block, and allowing a polymerization reaction to proceed, thereby retaining a gel-like material containing the probe in the hollow portion
(iv) Cutting the hollow fibers in a direction intersecting the longitudinal direction of the hollow fibers to form a block into thin pieces
The material used for the hollow fiber is not limited, and examples thereof include those described in, for example, Japanese patent application laid-open No. 2004-163211.
The hollow fibers are three-dimensionally arranged so that the lengths thereof in the longitudinal direction are the same (step (i)).
Examples of the alignment method include the following methods: a method in which a plurality of hollow fibers are arranged in parallel at predetermined intervals on a sheet-like material such as an adhesive sheet, and the sheet-like material is formed into a sheet shape, and then the sheet is wound into a spiral shape (see japanese patent application laid-open No. 11-108928); and (2) stacking 2 porous plates provided with a plurality of holes at predetermined intervals so that the holes are aligned, passing the hollow fibers through the holes, and then opening the interval between the 2 porous plates and temporarily fixing the porous plates, thereby filling the curable resin material in the periphery of the hollow fibers between the 2 porous plates and curing the material (see japanese patent application laid-open No. 2001-133453). The prepared array is embedded so as not to disturb the alignment (step (ii)).
As a method of embedding, in addition to a method of flowing a urethane resin, an epoxy resin, or the like into gaps between fibers, a method of bonding fibers to each other by thermal fusion bonding, or the like can be preferably cited.
The hollow portion of each hollow fiber is filled with a gel precursor polymerizable solution (gel-forming solution) containing an oligonucleotide probe, and a polymerization reaction is performed in the hollow portion (step (iii)). This makes it possible to hold the gel-like material to which the probe is fixed in the hollow portion of each hollow fiber. The gel precursor polymerizable solution is a solution containing a reactive substance such as a gel-forming polymerizable monomer, and is a solution which can be converted into a gel-like material by polymerizing and crosslinking the monomer or the like. Examples of such monomers include acrylamide, dimethylacrylamide, vinylpyrrolidone, and methylenebisacrylamide. In this case, a polymerization initiator or the like may be contained in the solution. After the probe is fixed in the hollow fiber, the block is cut in a direction intersecting with (preferably orthogonal to) the longitudinal direction of the hollow fiber to form a thin sheet (step (iv)). The thus obtained sheet can be used as a DNA chip. The thickness of the DNA chip is preferably about 0.01mm to 1 mm. The block can be cut by a slicer, a laser, or the like. As the above-mentioned fiber type DNA chip, for example, DNA chip (Genopal TM) manufactured by Mitsubishi chemical corporation, etc. can be preferably cited.
In the fiber-type DNA chip, the probes are arranged three-dimensionally in the gel as described above, and the three-dimensional structure can be maintained. Therefore, the detection efficiency is improved as compared with a flat DNA chip in which a probe is bound to a slide glass having a coated surface, and a highly reproducible examination can be performed with high sensitivity. The number of types of probes to be arranged on a DNA chip is preferably 500 or less, more preferably 250 or less, and still more preferably 100 or less for 1 DNA chip. By limiting the number (type) of the probes arranged in this way to a certain extent, it is possible to detect the target intraoral bacteria with higher sensitivity. The types of probes can be distinguished by their base sequences. Therefore, even if probes derived from the same gene are used, the probes are usually identified as other species as long as 1 base sequence is different.
3. Detection of bacterial genes in the oral cavity
In the method of the present invention, a method for detecting a gene of an oral bacterium in order to detect the bacterium is, for example, a method including the following steps.
(i) Extracting nucleic acid from an oral sample collected from a subject
(ii) Contacting the extracted nucleic acid with the oligonucleotide probe of the present invention or the DNA chip of the present invention
(iii) A step of calculating the SN ratio from the signal intensity obtained from the DNA chip, or a step of calculating the amount of bacteria
The details of the detection method will be described below according to the procedure.
(1) Concerning the step (i)
In this step, an oral sample collected from a subject or a test organism is used as a sample, and nucleic acids of bacteria contained in the sample are extracted. The type of the collected oral sample is not particularly limited. For example, saliva, plaque (subgingival plaque, supragingival plaque), tongue coating, mouth rinse, and the like can be used, and among these, plaque is preferable, and subgingival plaque collected from a site where periodontal disease bacteria inhabit most is more preferable.
The method for collecting the oral sample is not particularly limited, and may be appropriately selected depending on the type of the sample. For example, when saliva is used as an oral cavity sample, the following methods are exemplified: a method using a commercially available saliva collection kit, a method of collecting saliva contained in the mouth with a cotton swab, a method of directly collecting saliva into a container, and the like.
When plaque is used as an intraoral sample, the following methods can be mentioned: tooth surface brushing by a toothbrush, interdental brushing, tooth surface friction by a cotton swab, interdental friction by an interdental brush, a paper point (paper point) method, and the like. The tooth brush, cotton swab, interdental brush, or paper tip used for collecting plaque is immersed in sterile water and stirred as necessary, whereby the plaque is dissolved or suspended, and the resulting solution or suspension can be used as a specimen. The amount of collected plaque is not particularly limited, and may be, for example, 1 paper point. When the tongue coating is used as an intraoral sample, a method of rubbing the tongue surface with a cotton swab or the like may be mentioned. The cotton swab used for collecting the plaque is dissolved or suspended, and the obtained solution or suspension can be used as a sample. The amount of the collected tongue coating is not particularly limited, and may be, for example, 1 cotton bud.
When an oral cavity cleaning solution is used as an oral cavity sample, the following methods are exemplified: the oral cavity wash or water is contained in the mouth, saliva is collected in a container together with the oral cavity wash or water, and the obtained solution is used as a specimen. Examples of the oral cavity wash include sterilized physiological saline and the like. Subsequently, nucleic acid extraction of bacteria present in the obtained oral sample was performed. The method of extraction is not limited, and a known method can be used. Examples of the method include the following: an automatic extraction method using a facility, a method using a commercially available nucleic acid extraction kit, a method using bead disruption, a method using phenol extraction after proteinase K treatment, a method using chloroform, or a method of heating and dissolving a sample as a simple extraction method. They may also be processed in combination. In particular, the next step can be performed without extracting nucleic acids from the sample.
The nucleic acid obtained from the sample may be contacted with the DNA chip or the like as it is, or the amplified fragment may be contacted with the DNA chip or the like by amplifying a desired nucleotide sequence region by PCR or the like, without limitation. The region in which the obtained nucleic acid is amplified as a template is a portion of the nucleic acid region encoding the nucleotide sequence of the oligonucleotide disposed in the probe or DNA chip used in the present invention. The desired region to be amplified is not limited, and a plurality of types of mixtures can be amplified at once using the base sequence of a region highly conserved regardless of the type of the oral bacteria. The sequence to be used for such amplification can be determined by separating and purifying through experiments, analyzing the base sequence of the polynucleotide after separation, and determining based on the sequence, or by computer simulation (In Silico) by searching for a known base sequence using various databases such as base sequences and the like, and comparing the searched base sequences. The databases for nucleic acids and amino acids are not particularly limited, and examples thereof include the Japanese DNA database (DDBJ: DNA Data Bank of Japan), European Molecular Biology laboratory (EMBL: European Molecular Biology laboratory, EMBL nucleic acid sequence database (EMBL nucleic acid sequence Data library), Genetic sequence database (GenBank: Genetic sequence Data Bank), and the National Center for Biotechnology Information (NCBI: National Center for Biotechnology Information) classification database.
Specifically, the desired site for amplification is preferably the ribosomal RNA (16S rRNA) gene in the chromosomal DNA of the above-mentioned oral bacteria. Examples of PCR primers that can be used for amplifying this region include, for example, those shown in Table 2 (SEQ ID NOS: 37 and 38) and Table 3 (SEQ ID NOS: 41 to 53). The amplification of nucleic acid by the PCR method can be carried out according to a conventional method.
The nucleic acid and the amplified fragment thereof extracted in this step may be appropriately labeled and used in the detection process after hybridization. Specifically, it is possible to consider: a method of labeling the ends of PCR primers with various reporter dyes in advance, a method of introducing reactive nucleotide analogs at the time of reverse transcription reaction, a method of introducing biotin-labeled nucleotides, and the like. In addition, the labeling may be carried out by reacting with a fluorescent labeling reagent after the preparation. As the fluorescent reagent, for example, various reporter dyes (e.g., Cy5, Cy3, VIC, FAM, HEX, TET, fluorescein, FITC, TAMRA, Texas red, Yakima Yellow, etc.) can be used.
(2) Concerning the step (ii)
In this step, the nucleic acid or the amplified fragment thereof obtained in step (i) is brought into contact with the probe or DNA chip used in the present invention, specifically, a hybridization solution containing the nucleic acid or the like is prepared, and the nucleic acid or the like in the solution is bound (hybridized) to the oligonucleotide probe carried on the DNA chip. The hybridization solution can be appropriately prepared according to a conventional method using a buffer such as SDS or SSC. The hybridization reaction can be carried out under stringent conditions by appropriately setting reaction conditions (buffer type, pH, temperature, etc.) so that the nucleic acid or the like in the hybridization solution can hybridize to the oligonucleotide probe mounted on the DNA chip. The "stringent conditions" as used herein refer to conditions under which cross-hybridization with similar sequences is unlikely to occur or nucleic acids that are cross-hybridized with similar sequences are dissociated due to similar sequences, and specifically refer to conditions under which a DNA chip is washed during or after hybridization.
For example, as conditions for the hybridization reaction, the reaction temperature is preferably 35 to 70 ℃, more preferably 40 to 65 ℃, and the time for hybridization is preferably about 1 minute to 16 hours. As the conditions for washing the DNA chip after hybridization, the washing solution preferably has a composition of 0.24M Tris-HCl/0.24M NaCl/0.05% Tween-20, and the temperature at the time of washing is preferably 35 to 80 ℃ or 40 to 65 ℃, more preferably 45 to 60 ℃. More specifically, the salt (sodium) concentration is preferably 48 to 780mM and the temperature is preferably 37 to 80 ℃, and the salt concentration is more preferably 97.5 to 390mM and the temperature is preferably 45 to 60 ℃.
After washing, the detection intensity is measured for each spot by using a device capable of detecting a label such as a nucleic acid bound to the probe. For example, when the nucleic acid or the like is fluorescently labeled, the fluorescence intensity can be measured using various fluorescence detectors, for example, CRBIO (Hitachi Software Engineering), arrayWoRx (GE Healthcare), Affymetrix 428 Array Scanner (Affymetrix), GenePix (Axon Instruments), ScanArray (PerkinElmer), GenoPearlreader (Mitsubishi chemical Co., Ltd.), and the like. In these devices, for example, in the case of a fluorescence scanner, scanning can be performed by appropriately adjusting the output power of laser light and the sensitivity of a detection unit, and in the case of a CCD camera type scanner, scanning can be performed by appropriately adjusting the exposure time. The quantification method based on the scan results was performed using quantification software. The quantitative software is not particularly limited, and the quantitative determination may be performed by using an average value, a median value, or the like of the fluorescence intensity of the spot. In addition, in the quantitative determination, it is preferable to adjust the fluorescence intensity of the spot on which no probe is mounted as a background in consideration of the dimensional accuracy of the spot range of the DNA fragment and the like.
(3) Concerning the procedure (iii)
In this step, the bacterial count of the bacteria of the detection target bacterial species is calculated from the signal intensity obtained in the above-described step. For example, there is a method of expressing the ratio of the signal intensity of a probe for detecting a target bacterium to the signal intensity of the background in the form of an SN ratio. Since the signal intensity is proportional to the amount of bacteria present, the SN ratio can also be used directly for analysis without calculating the copy number.
Alternatively, the following method may also be used: a method of detecting each bacterium under a plurality of conditions while changing the concentration of chromosomal DNA of the bacterium, obtaining a conversion factor (calibration curve) calculated for each concentration of chromosomal DNA of each bacterium in advance based on the signal intensity obtained under each concentration condition, and calculating the concentration of chromosomal DNA from the signal intensity obtained under each condition, and the like. In this case, the results can also be calculated in the form of copy number of the bacteria.
In any case, the signal intensity and the copy number can be corrected by considering the correction coefficient for the signal intensity of the bacteria to be detected on each DNA chip. The order of correction and signal strength/copy number conversion is not limited.
4. Determination of periodontal disease status
In the present invention, the signal intensity of an intraoral bacterial population-derived nucleic acid present in an intraoral sample is measured, the proportion of the bacterial population present is calculated from the measured value of the signal intensity, and the state of periodontal disease is determined using the calculated value as an index.
The signal intensity of the nucleic acid derived from the intraoral bacterial group present in the intraoral sample can be measured using any instrument, and examples thereof include: the method using the DNA chip, the method using real-time PCR, and the method using FISH method described in the
The measured value of the signal intensity includes an SN ratio obtained from a DNA chip, a Ct value obtained by real-time PCR, fluorescence intensity obtained by FISH method, and the like.
The existence ratio of the bacterial group is a correlation between the bacterial amount of the bacterial species (positively related bacteria) that increases with an increase in the number of periodontal pockets and the bacterial amount of the bacterial species (negatively related bacteria) that decreases with an increase in the number of periodontal pockets.
Examples of the correlation include: the ratio of the total bacterial count of the positively correlated bacteria to the total bacterial count of the negatively correlated bacteria (the bacterial count of the Σ positively correlated bacteria/∑ negatively correlated bacteria), and a value obtained by subtracting the total bacterial count of the positively correlated bacteria from the total bacterial count of the negatively correlated bacteria (the bacterial count of Σ negatively correlated bacteria- ∑ positively correlated bacteria), a ratio of a value obtained by multiplying the total bacterial count of the positively relevant bacteria by a predetermined coefficient to a value obtained by multiplying the total bacterial count of the negatively relevant bacteria by a predetermined coefficient (sigma coefficient x bacterial count of the positively relevant bacteria/. sigma coefficient x bacterial count of the negatively relevant bacteria), a value of a sum of a value obtained by multiplying the total bacterial count of the positively relevant bacteria by a predetermined positive coefficient and a value obtained by multiplying the total bacterial count of the negatively relevant bacteria by a predetermined negative coefficient (sigma positive coefficient x bacterial count of the positively relevant bacteria +. sigma negative coefficient x bacterial count of the negatively relevant bacteria).
When the number of types of positively relevant bacteria is different from the number of types of negatively relevant bacteria, it is preferable to correct both of them so as to obtain a result calculated from the same number of types of bacteria.
For example, the average SN ratio of the "positively correlated bacteria" group is calculated by calculating the sum of the SN ratios of the "positively correlated bacteria" group and dividing the sum by the number of the types of the "positively correlated bacteria" group. Similarly, the average SN ratio of the "negative-related bacteria" population is calculated by summing up the SN ratios of the "negative-related bacteria" population and dividing the sum by the number of the types of the "negative-related bacteria" population.
Finally, the balance index can be obtained by obtaining the ratio of the average SN ratio of the positive correlation bacterium group to the average SN ratio of the negative correlation bacterium group.
As the existence ratio of the bacterial flora, it is preferable to use a "ratio" of the bacterial count of the bacterial species that increases with an increase in the numerical value of the periodontal pocket to the bacterial count of the bacterial species that decreases with an increase in the numerical value of the periodontal pocket.
The calculated value of the presence ratio thus obtained is referred to as an equilibrium index.
The numerator and denominator used for calculating the balance index are arbitrary, and either of them may be used as the denominator or the numerator. For example, the SN ratio of the bacterial species group decreasing with an increase in the value of the periodontal pocket may be taken as the denominator, the SN ratio of the bacterial species group increasing with an increase in the value of the periodontal pocket may be taken as the numerator, the SN ratio of the bacterial species group increasing with an increase in the value of the periodontal pocket may be taken as the denominator, and the SN ratio of the bacterial species group decreasing with an increase in the value of the periodontal pocket may be taken as the numerator.
In the conventional bacterial tests, the state of periodontal disease is determined by detecting bacteria corresponding to "harmful bacteria". Since they are bacterial species whose periodontal pockets increase to some extent, only deteriorated information can be obtained. In the present invention, the state of periodontal disease is determined by calculating the balance index using the bacterial group corresponding to "beneficial bacteria", and therefore, the state of health can be determined.
To explain in more detail, consider the following.
The amount of bacteria of "harmful bacteria" is a monotonous increasing function with respect to the value of periodontal pocket, and the amount of bacteria of "beneficial bacteria" is a monotonous decreasing function with respect to the value of periodontal pocket.
When the amount of bacteria of "harmful bacteria" is taken on the vertical axis and the numerical value of the periodontal pocket is taken on the horizontal axis, the numerical value of the periodontal pocket may not be a determinable value between 0 and 3 mm.
On the other hand, it is excellent in that when the index of the bacterial count of "harmful bacteria"/"beneficial bacteria" is taken as the vertical axis and the numerical value of periodontal pocket is taken as the horizontal axis, the function clearly shows an inflection point and can be determined in the vicinity thereof. Moreover, the number of periodontal pockets is a value that can be determined between 0 and 3mm, and the health status can be determined using this value.
In the present invention, it is preferable to determine the state of periodontal disease by comparing the balance index with a cutoff value.
The cutoff value is a value having a function as a threshold value or a reference value of the existence ratio (balance index) of the bacterial population.
The signal intensity of the nucleic acid derived from the intraoral bacteria group present in the intraoral sample can be determined by preparing a standard measurement, calculating the proportion of the bacteria group present from the measured value of the signal intensity, plotting a ROC curve from the calculated value (balance index), and determining the ROC curve. The cut-off value is preferably chosen in such a way that the distance to the upper left of the ROC graph is reduced. However, the sensitivity can be appropriately changed depending on the purpose (the desired sensitivity and the desired specificity).
In addition, the cutoff value may be determined by cluster analysis in addition to the ROC curve described above. More specifically, it is possible to consider: in the case of cluster analysis using the k-means Method, an optimal number of clusters is studied and determined by the Elbow Method, or after the number of clusters is automatically output using the x-means Method, etc., an index corresponding to a boundary between the clusters is used as a cutoff value.
As a first mode of the determination model creation, a determination model based on the existence ratio (balance index) of "the bacterial species increasing with the increase in the numerical value of the periodontal pocket" and "the bacterial species decreasing with the increase in the numerical value of the periodontal pocket" can be considered.
Examples of various bacteria are described in
The bacterial species that increase with an increase in the number of periodontal pockets are not particularly limited, and preferably include 1 or more bacterial species other than the fusobacterium nucleatum species ("disease progression indicator bacteria"). Specifically, more than 1 kind selected from Porphyromonas gingivalis, Stannia forskoensis, Treponema denticola, Campylobacter tenuis, Campylobacter rectus, Showa Campylobacter, Clostridium paradenticola, Prevotella intermedia, Streptococcus astrus, Actinomyces aggregatus, Exosella denticola, Bordetella expectorata, Bordetella intermedia, Eubacterium crypthecium, Eubacterium tangling, Porphyromonas pulposus, Protozoa gingivalis, Streptococcus gastris, and Treponema sojae is are preferable, and more preferable from Porphyromonas gingivalis, Stannia fortunata, Treponema denticola, Campylobacter rectus, Campylobacter showa, Clostridium paradenticola, Streptococcus astrus, Actinomyces aggregatibacter, Exonella denticola, Bordetella expectorata, Bordetella intermedia, Eubacterium crypticans, Campylobacter xylinum, Campylobacter palmita-checking, and Campylobacter, More than 1 of eubacterium tangling, porphyromonas endodontium, and gingival sulcus production line bacteria.
The kind of the bacterium to be used is preferably 4 or more, more preferably 8 or more, further preferably 12 or more, and particularly preferably 14 or more. The number of the bacterial species used is preferably 100 or less, more preferably 75 or less, still more preferably 50 or less, and particularly preferably 25 or less.
The type of bacteria that decreases with an increase in the number of the peripheral bags is not particularly limited, and specifically, is preferably selected from the group consisting of Streptococcus parahaemolyticus, Haemophilus parainfluenzae, Streptococcus salivarius, Pediococcus brevicella, Rostellularia carinatus, Prevotella pseudopratensis (A.rava, OT308), Leronella rosenbergii, Porphyromonas pasteurii, Prevotella charitis, Prevotella pallida, atypical Veillonella, Actinomyces Geranii, megasphaerobacter parvum, Prevotella loensis, Neisseria flavus, Microbacterium morganii, Porphyromonas catori, Roselleri, Corynebacterium malassecola, Eubacterium coulis, twin haemolyticus, Prevotella melanogenesis, Prevotella denticola, Prevotella blackcurriculorum, Campylobacter, caphilus gingivalis, carbon dioxide Cellulosiphilus flavus, carbon dioxide Cellulus, and Bacillus flavivirus, More preferably at least 1 of the group consisting of Streptococcus paracasei, Haemophilus parainfluenzae, Streptococcus intermedius, Streptococcus mitis, Streptococcus pauciflorus bv2, Actinomyces naeslundii II, harmful Pseudomonas lunata, SR1 sp.OT 345, Micromonospora parvum, Streptococcus sorbinus, Actinomyces tundiformis and Prevotella histophila, and more preferably at least one of the group consisting of Streptococcus parahaemolyticus, Haemophilus parainfluenzae, Streptococcus salivarius, Streptococcus parvum, Rostella carinii, Prevotella pseudopraerula (A. rava, OT308), Leptococcus rouxii, Porphyromonae, Prevotella destructor, Prevotella charitis, Prevotella vulgaris, Prevotella pallidum, Pasteur, Salmonella typica, Actinomyces kuneferi, Microbacterium amycoltsi, Prevotella rosenbergii, Streptococcus parvum, Porphyrae, Porphyra, Porphyrom, Corynebacterium equisimilis, Eubacterium nidulans, twin hemolytic coccus, Prevotella melanogenesis, Prevotella denticola, Prevotella melanogaster, Campylobacter succinogenes, capnocytophaga gingivalis, capnocytophaga lutescens, capnocytophaga sputigena, Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis bv2, Actinomyces carinatus, Microveillonella parvula, Actinomyces naeslundii II and more than 1 of harmful Paeonia lunata.
The kind of the bacterium to be used is preferably 2 or more, more preferably 10 or more, and still more preferably 20 or more. The number of the bacterial species used is preferably 100 or less, more preferably 75 or less, still more preferably 50 or less, and particularly preferably 25 or less.
The measurement value of the signal intensity of the nucleic acid derived from the bacteria present in the oral cavity sample is determined based on the known periodontal disease state, the proportion of the bacteria present is calculated, and the cutoff value is determined from the calculated value (balance index).
Then, in the determination of a sample in which the periodontal disease state is unknown, the bacteria group is detected all at once, and the balance index is calculated in the same manner and compared with the cutoff value, thereby determining the state.
According to the determination model of the first aspect, samples whose periodontal disease state is unknown can be determined as 2 groups from the cutoff value.
As an example, a model for determining a non-disease state and a disease state will be described.
The non-diseased state and the diseased state may be defined as appropriate, and in the present application, the non-diseased state is defined as a periodontal pocket depth of 1 to 3mm, and the diseased state is defined as a periodontal pocket depth of 5mm or more. That is, the periodontal pocket depth of 4mm is an undefined state of a diseased state or a non-diseased state.
The signal intensity of nucleic acids derived from various bacterial groups is measured for samples having a periodontal pocket depth of 1 to 3mm and samples having a periodontal pocket depth of 5mm or more, the ratio of the presence of various bacterial groups is calculated from the measured values, and the cutoff value is determined from the calculated value (balance index). Then, for the sample with the periodontal pocket depth of 4mm, the balance index is calculated in the same manner, and by comparing this with the previous cutoff value, the group with the periodontal pocket depth of 4mm whose disease state is unknown can be determined as a non-disease state (the same level as the group with the periodontal pocket depth of 1 to 3 mm) and a disease state (the same level as the group with the periodontal pocket depth of 5 mm). The method of the present invention is very useful in that it enables determination of a 4mm group of periodontal pocket depths, which are difficult to determine at present.
As a second mode of the determination model creation, a determination model based on the existence ratio of "disease progression index bacteria (fusobacterium nucleatum species)" and "bacterial species decreasing with an increase in the number of periodontal pockets" may be considered. The group of "bacterial species increasing with an increase in the number of periodontal pockets" in the first embodiment was replaced with a Fusobacterium nucleatum species as the "disease progression indicator bacteria".
Examples of various bacteria are described in
The Fusobacterium nucleatum species is not particularly limited, and specifically, it is preferably 1 or more species selected from the group consisting of Fusobacterium nucleatum subspecies, Fusobacterium nucleatum Venezueli subspecies and Fusobacterium nucleatum pleomorphe subspecies, and more preferably 1 or 2 species selected from the group consisting of Fusobacterium nucleatum subspecies and Fusobacterium nucleatum subspecies.
As the bacterial species that decrease with an increase in the number of the peripheric sacks, the same bacterial species as in the first embodiment can be preferably used.
The cutoff value can be calculated in the same manner as in the determination model of the first embodiment, and thus the determination can be made as 2 groups.
According to the judgment model of the second aspect, a state in which the number of periodontal pockets is small can be obtained more favorably than in the first aspect.
As an index similar to the present invention, there is an example in which a ratio of the total bacterial amount of porphyromonas gingivalis, bacteroides forsteriae, and treponema denticola as "harmful bacteria" to the bacterial amount of fusobacterium nucleatum as "disease course index bacteria" is used as an index. In the present invention, the ratio to the "beneficial bacteria" group is different from the "beneficial bacteria" group as an indicator. According to the index of the present invention, the progress of deterioration can be determined more clearly.
To explain in more detail, consider the following.
The amount of bacteria of "harmful bacteria" is a monotonous increasing function with respect to the value of periodontal pocket, and the amount of bacteria of "beneficial bacteria" is a monotonous decreasing function with respect to the value of periodontal pocket
When the index of the bacterial count of "harmful bacteria"/"bacterial count of disease index bacteria" is taken on the vertical axis and the numerical value of periodontal pocket is taken on the horizontal axis, the "harmful bacteria" in the healthy sample is small, and the result is not detected at all. That is, the number of periodontal pockets may not be a value that can be determined between 0 and 3 mm.
On the other hand, it is excellent in that when the index of the bacterial count of "harmful bacteria", "disease progression index bacteria" and the bacterial count of "beneficial bacteria" is taken on the vertical axis and the numerical value of periodontal pocket is taken on the horizontal axis, the function clearly shows an inflection point and can be determined in the vicinity thereof. Moreover, the number of periodontal pockets is a value that can be determined between 0 and 3mm, and the health status can be determined using this value.
In the present invention, the ratio of the bacteria may be used in combination with the following (a) and (b).
(a) Correlation between the amount of bacteria of the bacterial species (including 1 or more species of bacteria other than F. nucleatum species) that increase with an increase in the numerical value of periodontal pocket and the amount of bacteria of the bacterial species that decrease with an increase in the numerical value of periodontal pocket
(b) Correlation between the amount of bacteria of Fusobacterium nucleatum species and the species of bacteria that decrease with increasing number of periodontal pockets
When a DNA chip is used, since a plurality of bacteria groups can be detected all at once, a plurality of balance indices can be calculated at the same time. Therefore, it is also possible to simultaneously determine 2 balance indexes as axes, and to classify the indexes into groups of 2 × 2 to 4.
The determination of the state of periodontal disease obtained by the present invention is merely a determination in which the state is estimated from the number of bacteria or an SN ratio proportional to the amount of bacteria, and does not indicate an accurate disease state. That is, an accurate diagnosis of the condition requires a diagnosis by the dentist. However, according to the present invention, since the determination can be made independently of the numerical value of the periodontal pocket, it is possible to perform early detection and early treatment of periodontal disease from a novel viewpoint and also contribute to prevention thereof.
5. Determination of therapeutic Effect of periodontal disease
The treatment is a treatment usually performed by a dentist and a dental care professional at the front of the dentist, and examples of the basic periodontal treatment include plaque control (tooth brushing instruction), removal of dental plaque (Scaling and odontolith), and adjustment of occlusion. Further, there is exemplified surgical treatment performed when, as a result of reevaluation examination after the primary periodontal treatment, tartar has entered a deep portion of the pocket and is not removed and not cured. Specific examples of the surgical treatment include flap surgery, periodontal tissue regeneration therapy, and plastic surgery (periodontal surgery). In addition, "periodontal support therapy (SPT)" which is continuous professional care after completion of periodontal therapy is also important as an indispensable therapy for maintaining "stable disease" and maintaining good prognosis of periodontal therapy.
The treatment effect of periodontal disease was determined by collecting samples before and after the treatment of periodontal disease and comparing the data.
As the most basic idea, the treatment effect can be objectively determined by making full use of clinical information of a sample and clarifying bacteria that increase and decrease before and after treatment. In addition, by using the data of bacteria after treatment, bacteria that are not easily reduced by treatment can be clarified, and specific treatment can be performed.
According to the method of the present invention, the effect of periodontal disease treatment can be determined from the balance index of a plurality of bacteria by performing the determination described in "4. determination of periodontal disease state" above before and after treatment. In particular, the state of periodontal disease showing the same pocket value can be further classified into 4 categories.
When the determination is made in consideration of the numerical information of the periodontal pocket, the determination of stable disease state can be made. For example, even if the number of periodontal pockets is 4mm or more, if the balance index described in "4. determination of the state of periodontal disease" is determined as "mild", the condition can be considered to be stable. On the other hand, even if the value of the periodontal pocket is 3mm or less, if the balance index is determined to be "severe", it can be determined that there is a possibility that the treatment is considered.
In the present description, the SN ratio of the DNA chip has been described as the measurement value indicating the bacterial load, but the measurement value is included in the scope of the present invention as long as the measurement value can be used as a numerical value having the same meaning as the SN ratio of the DNA chip. For example, the copy number of bacteria obtained by conversion from the SN ratio of a DNA chip, the copy number of bacteria obtained by real-time PCR quantification, Ct value indicating the degree of quantity, the read number obtained from the result of a next-generation sequencer, the relative quantity percentage converted from the read number, and the like can be considered.
6. Oligonucleotide probe set
The present invention provides an oligonucleotide probe set for oral bacteria detection, which comprises the DNA of the following (a) or (b).
(a) DNA comprising base sequences represented by SEQ ID Nos. 1 to 33
(b) DNA having 90% or more identity to the base sequence represented by SEQ ID Nos. 1 to 33 and hybridizing with a part of the base sequence of the 16SrRNA gene or the complementary strand thereof in the chromosomal DNA of oral bacteria
The probe used in the present invention may be DNA composed of any combination of DNA having 33 kinds of base sequences represented by SEQ ID Nos. 1 to 33. For example, the DNA may be any 1 kind of DNA among DNAs consisting of the base sequences represented by SEQ ID Nos. 1 to 33, or may be a combination of 2 kinds of DNA, or may be a combination of 32 kinds of DNA, or may be a combination of 33 kinds of DNA.
The stringent conditions for the "hybridization" are the same as those described above.
Further, as the oral bacteria to be detected, there may be mentioned at least one type of bacteria belonging to any of the following genera: porphyromonas, tania, treponema, prevotella, campylobacter, clostridium, streptococcus, reuterium, capnocytophaga, elkessella, actinomyces, veillonella and selenomonas.
The present invention also provides a microarray for detecting oral bacteria, which is provided with the oligonucleotide probe set. In the present invention, as the microarray, the microarray described in "2. DNA chip for oral bacterial gene detection used for measuring the amount of oral bacteria" can be used.
The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
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