Patent Application
20220205011
The present invention relates to a method for detecting periodontopathic bacteria, and more specifically to a method for detecting periodontopathic bacteria which does not require special equipment, allowing periodontopathic bacteria that are measured under high temperature conditions, to be detected at room temperature in less than 10 minutes (about 2 to 8 minutes).
It is said that approximately 80% of adults in Japan are affected by periodontal disease. Periodontal disease causes tooth loss and it has become clear that, in accompaniment, not only are dysgeusia and abnormal salivation caused but abnormalities of the central nervous system and the autonomic nervous system are also caused. Further in recent years, it has come to be noted that periodontal disease is a risk factor of various systemic disorders, such as coronary heart disease, cerebral infarction, etc.
Methods for inspecting bacterial infection and inflammation, among inspection items of periodontal disease, include a method of visually judging stained tooth surfaces using a plaque staining solution, plaque adhesion condition inspection, with which staining is not performed and tooth surfaces are abraded with a tip of dental probe or dental explorer, etc., to judge whether or not plaque is adhered, periodontopathic bacteria inspection, with which subgingival plaque is sampled with a paper point and an inspection agency is requested to measure bacterial count by a DNA quantitation method, etc., an antibody inspection method, with which an IgG antibody titer in serum is measured for periodontopathic bacteria, etc. These methods not only require time, labor, and cost but also require special facilities and skills and therefore do not enable a plurality of patients to be inspected at the same time in a simple and rapid manner and the burden on a patient is also large.
Among periodontopathic bacteria, the three types of bacteria, Porphyromonas gingivalis (P. g), Treponema denticola (T. d), and Tannerella forsythia (T. f) are referred to as the red complex, which is considered to be the most dangerous bacterial types group that causes severe periodontal disease. These three types of bacteria have arginine-specific peptidase activity (trypsin-like enzymatic activity) and methods of detecting trypsin-like enzymatic activity using a synthetic substrate are reported in Patent Documents 1 to 4.
Also, it is known that this enzyme activity is activated by a reducing agent (Patent Documents 3 and 4, Non-Patent Document 1). It has also been reported that the enzyme activity can be analyzed at room temperature in 10 minutes. (Patent Document 4).
If the trypsin-like enzymatic activity can be analyzed and judged in a shorter time and a simpler manner for the presence of the three important types of bacteria among periodontopathic bacteria, this would be a useful method for screening for periodontal disease.
However, with trypsin-like enzymatic activity, sufficient sensitivity cannot be obtained other than at its optimal temperature of 50 to 60° C. and for measurement, special equipment capable of maintaining the optimal temperature is necessary and this is an obstacle to dissemination of inspection of periodontopathic bacteria. Analyzing enzyme activity after a period of 10 minutes at room temperature also lengthens the time constraint for the patient. It has therefore been necessary to immediately detect periodontopathic bacteria at locations where samples are sampled from patients.
The present inventors, focusing on the chemical properties of P.g, T.d and T.f, have found that if a compound having an antioxidant effect or a compound having the action of protecting SH groups (mercapto groups) and cleaving disulfide bonds is present when analyzing trypsin-like enzymatic activity, then it is possible to analyze trypsin-like enzymatic activity with high sensitivity at room temperature in less than 10 minutes, and have thereupon completed the present invention.
It is an object of the present invention to provide a method of analyzing periodontopathic bacteria that does not require special equipment, and that allows trypsin-like enzymatic activity specific to three types of periodontopathic bacteria, P.g, T.d and T.f, which are measured under high temperature conditions, to be analyzed with high sensitivity at room temperature in less than 10 minutes (about 2 to 8 minutes).
The invention according to claim 1 is a method for detecting periodontopathic bacteria in which periodontopathic bacteria are detected in a sample at room temperature and in less than 10 minutes, whereby a first compound having an antioxidant effect and/or a second compound having action of protecting SH groups and cleaving disulfide bonds, is contacted with the sample together with N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride at room temperature and the mixture is allowed to stand at room temperature for less than 10 minutes, to allow detection of periodontopathic bacteria in subsequent coloration analysis with a coloring reagent, wherein the first compound is at least one selected from among L-ascorbic acid, L-cysteine hydrochloride, and glutathione, and the second compound is at least one selected from among DTT, thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP.
The invention according to claim 2 is a method for detecting periodontopathic bacteria in which periodontopathic bacteria are detected in a sample at room temperature and in less than 10 minutes, whereby a sample containing a first compound having an antioxidant effect and/or a second compound having action of protecting SH groups and cleaving disulfide bonds, is added to N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride and the mixture is allowed to stand at room temperature for less than 10 minutes, to allow detection of periodontopathic bacteria in subsequent coloration analysis with a coloring reagent, wherein the first compound is at least one selected from among L-ascorbic acid, L-cysteine hydrochloride, and glutathione, and the second compound is at least one selected from among DTT, thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP.
The invention according to claim 3 is a method for detecting periodontopathic bacteria in which periodontopathic bacteria are detected in a sample at room temperature and in less than 10 minutes, whereby a first compound having an antioxidant effect and a second compound having action of protecting SH groups and cleaving disulfide bonds are mixed and added to a water absorbent material together with N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride and the sample is contacted with the water absorbent material, and then allowed to stand at room temperature for less than 10 minutes, to allow detection of periodontopathic bacteria in subsequent coloration analysis with a coloring reagent, wherein the first compound is at least one selected from among L-ascorbic acid, L-cysteine hydrochloride, and glutathione, and the second compound is at least one selected from among DTT, thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP.
The invention according to claim 4 is a method for detecting periodontopathic bacteria in which periodontopathic bacteria are detected in a sample at room temperature and in less than 10 minutes, whereby after a first compound having an antioxidant effect and a second compound having action of protecting SH groups and cleaving disulfide bonds are mixed, the mixture is added to the sample, and then N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride is added to the sample and the mixture is allowed to stand at room temperature for less than 10 minutes, to allow detection of periodontopathic bacteria in subsequent coloration analysis with a coloring reagent, wherein the first compound is at least one selected from among L-ascorbic acid, L-cysteine hydrochloride, and glutathione, and the second compound is at least one selected from among DTT, thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP.
The first compound is a chemical substance having an antioxidant effect, and it is selected from among L-ascorbic acid, L-cysteine hydrochloride, and glutathione. The first compound may be a single type or a combination of more than one type.
The second compound is a chemical substance having action of protecting SH groups and cleaving disulfide bonds, and it is selected from among DTT (dithiothreitol), thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP (tris(2-carboxyethyl)phosphine hydrochloride). The second compound may be of one type or a plurality of types may also be combined.
In the analysis of the trypsin-like protease activity, one of either of the first compound and the second compound may be present or both the first compound and the second compound may be mixed.
Room temperature indicates a state of not being heated or cooled from an external system. It is approximately 20° C. to 30° C.
The sample may be any sample to be analyzed such as an oral cavity swab sample, plaque sample, tongue swab or tongue coating sample or saliva, or a swab from an implant, bridge or denture removed from the mouth, in the form of a solution diluted with an appropriate medium or an extract liquid obtained by extraction of periodontopathic bacteria from the sample to be analyzed using an appropriate medium.
The first compound and/or the second compound may be incorporated, together with the substrate, in a water absorbent material (dry retention method). The substrate used is N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride, a substance that causes chemical reaction when acted upon by an enzyme of a periodontopathic bacterium contained in a sample. The reagent is incorporated in the water absorbent material (test specimen or carrier), the analysis object sample and the water absorbent material are put in direct contact, and presence or non-presence of a coloring substance that is released by enzyme activity of the periodontopathic bacteria is analyzed. In this case, a coloring reagent may be used as necessary.
As the coloring reagent, any of various known reagents may be used. For example, sodium 4-hydroxy-3-[(2,4-dihydroxy-3-quinolyl)azo]benzenesulfonate, 4-benzoylamino-2,5-diethoxybenzenediazonium, 4-(dimethylamino)cinnamaldehyde, etc., may be used.
The water absorbent material is not restricted in particular as long as it is a carrier having a water absorbing property and capable of incorporating the first compound and/or the second compound. Paper, filter paper, cellulose, nonwoven fabric, glass fiber, porous filter, or cotton, etc., can be cited as examples. The substrate may also be incorporated in the water absorbent material. The water absorbent material may be used as it is or may be provided on a suitable member, for example, waterproof paper, glass, plastic, wood, or metal, etc., to facilitate handling. Shape, length, and thickness of the water absorbent material are not restricted in particular as long as the sample can be contacted.
As the dry retention method, the first compound and/or the second compound may be dissolved or dispersed in a suitable solvent together with or separately of the substrate and the solution (or dispersion) may be incorporated in the water absorbent material. As the solvent, for example, tris-hydrochloride buffer solution, tris-maleate buffer solution, phosphate buffer solution, glycine buffer solution, sodium borate buffer solution, carbonate-bicarbonate buffer solution, Good's buffer solution, or purified water, etc., may be used. Further, an abovementioned solvent added with a suitable surfactant, for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenol ethoxylate, etc., may be used.
The water absorbent material may be impregnated with 10 μL to 500 μL of a 1 mM to 100 mM solution (dispersion) of the first compound and/or the second compound and thereafter used upon drying by air drying, blow drying, depressurized vacuum drying, or freeze drying.
The first compound and/or the second compound may be dissolved in a suitable solvent and thereafter added to the sample (solution method). Also, the first compound and/or the second compound may be added directly to the sample.
As the solvent, for example, tris-hydrochloride buffer solution, tris-maleate buffer solution, phosphate buffer solution, glycine buffer solution, sodium borate buffer solution, carbonate-bicarbonate buffer solution, Good's buffer solution, or purified water, etc., may be used. Further, an abovementioned solvent added with a suitable surfactant, for example, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, octylphenol ethoxylate, etc., may be used.
A 10 mM to 1000 mM solution of the first compound and/or the second compound of 1/10 volume of the volume of the sample may be added to the sample. Also, if the first compound and/or the second compound are or is to be added directly to the sample, the addition amount is approximately 1 mM to 100 mM.
The invention according to claim 5 is the method for detecting periodontopathic bacteria according to any one of claims 1 to 4, wherein the concentration of the first compound is 6.25 mM to 100 mM.
The invention according to claim 6 is the method for detecting periodontopathic bacteria according to any one of claims 1 to 5, wherein the concentration of the second compound (excluding TCEP) is 6.25 mM to 100 mM.
The invention according to claim 7 is the method for detecting periodontopathic bacteria according to any one of claims 1 to 6, wherein the concentration of the TCEP is 6.25 mM to 25 mM.
If the concentration of the first compound is in the range of 6.25 mM to 100 mM, the second compound is DTT, thioglycolic acid, thioglycerol, or mercaptoethanol and its concentration is in the range of 6.25 mM to 100 mM, or the second compound is TCEP and its concentration is 6.25 mM to 25 mM, it will be possible to detect a periodontopathic bacterium in about 2 minutes. It is therefore possible to analyze and judge the presence of periodontopathic bacteria in a very short time, which is highly effective for screening of periodontal disease.
According to the present invention, a first compound having an antioxidant effect or a second compound having action of protecting SH groups and cleaving disulfide bonds is added to a sample, whereby trypsin-like enzymatic activity specific to the three types of bacteria P.g, T.d and T.f, which has conventionally been measured under high temperature conditions, can be analyzed at room temperature in less than 10 minutes (about 2 to 8 minutes). This allows convenient analysis and determination of trypsin-like enzymatic activity in a short period of time, which is effective for screening of periodontal disease.
Although examples of the present invention shall now be described specifically, these examples do not limit the scope of the present invention.
(Preparation of First Compounds and Second Compounds)
As compounds (first compounds) having an antioxidant effect, 62.5 mM, 125 mM, 250 mM, 500 mM, and 1000 mM of each of L-ascorbic acid, L-cysteine hydrochloride, and glutathione were sampled, and each of the sampled first compounds was dissolved in 50 mM tris-maleate buffer solution pH 8.5 and thereafter, pH adjustment was performed such that the pH became 8.5.
As compounds (second compounds) having action of protecting SH groups and cleaving disulfide bonds, each of DTT, thioglycolic acid, thioglycerol, mercaptoethanol, and TCEP were dissolved in 50 mM tris-maleate buffer solution pH 8.5 to concentrations of 62.6 mM, 125 mM, 250 mM, 500 mM, and 1000 mM and thereafter, pH adjustment was performed such that the pH became 8.5.
(Preparation of Substrate)
As a substrate, N-α-benzoyl-DL-arginine-2-naphthylamide hydrochloride (obtained from Sigma-Aldrich Japan LLC) was dissolved in 50 mM tris-maleate buffer solution pH 8.5. The substrate concentration is 0.11% by weight.
(Preparation of Coloring Solution)
4-(dimethylamino) cinnamaldehyde (DMAC) (obtained from Sigma-Aldrich Japan LLC) was dissolved in 1 mol/L hydrochloric acid. The concentration of DMAC is 0.1% by weight.
(Culturing of P. g bacteria (sample))
Prepared Porphyromonas gingivalis (P. g bacteria) JCM12257 was inoculated into a broth medium and anaerobically cultured at 37° C. for 24 hours and a bacterial culture solution of 1.0×107 cfu/mL was obtained.
The broth medium was prepared with 3.0 g of trypticase soy broth, 0.5 g of yeast extract, 0.05 g of L-cysteine hydrochloride, 0.1 mL of hemin solution, 0.02 mL of vitamin K1 solution, and 100 mL of distilled water and after preparation, sterilization at 121° C. was performed for 15 minutes.
With the hemin solution, 0.005 g of hemin and 0.0174 g of potassium hydrogen phosphate were dissolved in 1 mL of distilled water. With the vitamin K1 solution, 5 mg of vitamin K1 were dissolved in 1 mL of ethanol.
As a method for measuring bacterial count, the broth culture solution after anaerobic culturing was 10-fold serially diluted with sterilized physiological saline and inoculated onto CDC anaerobe sheep blood agar (obtained from Becton, Dickinson and Co.) and thereafter, anaerobic culturing at 37° C. was performed for 48 hours and bacterial count measurement was performed by counting colonies grown on the medium with the naked eye.
(A) Dry Retention of Substrate and First Compound or Second Compound
A prepared first compound or second compound and the substrate were mixed at a ratio of 1:9, and 10 mm paper disks (obtained from Advantec Co., Ltd.) were impregnated with 80 μL of the mixture per disk. The paper disks were then dried overnight at 25° C.
(B) Preparation of P. g
The bacterial culture solution after 24 hours of culturing was 10-fold serially diluted with 50 mM tris-maleate buffer solution pH 8.5 to prepare bacterial suspensions of 1.0×106 cfu/mL, 1.0×105 cfu/mL, 1.0×104 cfu/mL, and 1.0×103 cfu/mL.
(C) Test Method
Two of the paper disks, impregnated with the first compound or the second compound of each concentration and the substrate and then each impregnated with 80 μL of the prepared P. g bacterial suspension, were prepared and left to stand respectively for 2 minutes, 5 minutes, 8 minutes, 10 minutes, 15 minutes, and 20 minutes, one at room temperature and the other at 50° C. The former shall be referred to as room temperature analysis and the latter as 50° C. analysis. Thereafter, 30 μL of the coloring solution was dripped onto each paper disk and coloration of each paper disk was observed and judged with the naked eye.
(D) Results
When an enzyme reaction is carried out with a sufficient amount of substrate concentration, the reaction rate changes depending on the concentration of the detection substance, with a higher detection substance concentration allowing the reaction to reach a steady state in a shorter time, and conversely with a lower detection substance concentration requiring more time for the reaction to reach a steady state. In the presence of the first compound and second compound, however, as shown in
(A) Dry Retention of Substrate
The prepared substrate was mixed in 50 mM tris-maleate buffer solution pH 8.5 such that a volume ratio would be 9:1 and 10 mm paper disks (obtained from Advantec Co., Ltd.) were impregnated with 80 μL of the mixture per disk. The paper disks were then dried overnight at 25° C.
(B) Preparation of P.g Bacteria
The prepared first compound or second compound was diluted 9-fold with 50 mM tris-maleate buffer solution pH 8.5 to obtain a diluted solution of the compound.
The bacterial culture solution after 24 hours of culturing was 10-fold serially diluted with the diluted compound solutions to prepare bacterial suspensions of 1.0×106 cfu/mL, 1.0×105 cfu/mL, 1.0×104 cfu/mL, and 1.0×103 cfu/mL.
(C) Test Method
Two of the paper disks, impregnated with the substrate and then impregnated with 80 μL of the prepared P. g bacterial suspension, were prepared and left to stand respectively for 2 minutes, 5 minutes, 8 minutes, 10 minutes, 15 minutes, and 20 minutes, one at room temperature and the other at 50° C. The former shall be referred to as room temperature analysis and the latter as 50° C. analysis. Thereafter, 30 μL of the coloring solution was dripped onto each paper disk and coloration of each paper disk was observed and judged with the naked eye.
(D) Results
Similar to the results for the dry retention method in Example 1, it was shown that in the presence of a first compound or second compound, sensitivity obtained with 50° C. analysis is obtained in an equivalent manner at a reaction time of less than 10 minutes, even at room temperature. In other words, it was shown that a difference in sensitivity of 100-fold was found between room temperature analysis and 50° C. analysis in the absence of a first compound or second compound, with the minimum bacterial concentration (P.g at 1×104 cfu/mL) being undetectable even at 20 minutes, but that in the presence of a first compound or second compound, with any of the compounds, even with room temperature analysis it is possible to detect the minimum bacterial concentration in a manner equivalent to 50° C. analysis in less than 10 minutes, though with some differences in minimum analysis time depending on the type of compound. Table 2 shows a comparison between minimum analysis times allowing detection of the minimum bacterial concentration in the presence of different concentrations of first compounds and second compounds, for room temperature analysis and 50° C. analysis. As shown in Table 2, it was confirmed that the effects of the first compound and second compound had wide optimal concentrations, though with differences in minimum analysis time depending on the types and concentrations of the compounds, and detection of the minimum bacterial concentration was possible in less than 10 minutes at room temperature. With thioglycolic acid and TCEP, however, while it was possible to detect the minimum bacterial concentration in less than 10 minutes by the dry retention method in Example 1, detection was not possible by the solution method. This is believed to be due to the effect of the second compound being added directly to the bacterial culture solution.
It has become clear that while sufficient sensitivity cannot be obtained in room temperature analysis for enzyme activity having an optimal temperature of 50 to 60° C., the present invention enables, by dry retention in a water absorbent material or addition to an sample of a first compound having an antioxidant effect or a compound having action of protecting SH groups and cleaving disulfide bonds, analyzing the enzyme activity in less than 10 minutes at a sensitivity equivalent to 50° C. analysis even with room temperature analysis.
(A) Mixed Preparations of First Compounds and Second Compounds
The first compounds and the second compounds were mixed in combinations shown in Table 3. Mixing concentrations were adjusted by dissolving 500 mM of each of a first compound and a second compound in 50 mM tris-maleate buffer solution pH 8.5 and thereafter mixing equivalent amounts of the first compound and the second compound such that the concentration of each would be 250 mM.
(B) Dry Retention of Substrate and Mixture of First Compound and Second Compound
A solution with a first compound and a second compound mixed and the prepared substrate were mixed at a ratio of 1:9, and 10 mm paper disks (obtained from Advantec Co., Ltd.) were impregnated with 80 μL of the mixture per disk. The paper disks were then dried overnight at 25° C. (Final concentration of each of the first compound and the second compound mixed was 25 mM.)
(C) Preparation of P. g Bacteria
The bacterial culture solution after 24 hours of culturing was 10-fold serially diluted with 50 mM tris-maleate buffer solution pH 8.5 to prepare bacterial suspensions of 1.0×106 cfu/mL, 1.0×105 cfu/mL, 1.0×104 cfu/mL, and 1.0×103 cfu/mL.
(D) Test Method
Two of the paper disks, impregnated with the mixture of the first compound and the second compound and the substrate and then each impregnated with 80 μL of the prepared P. g bacterial suspension, were prepared and left to stand respectively for 2 minutes, 5 minutes, 8 minutes, 10 minutes, 15 minutes, and 20 minutes, one at room temperature and the other at 50° C. The former shall be referred to as room temperature analysis and the latter as 50° C. analysis. Thereafter, 30 μL of the coloring solution was dripped onto each paper disk and coloration of each paper disk was observed and judged with the naked eye.
(E) Results
Table 4 shows minimum analysis times at which minimum bacterial concentrations could be detected with combinations of added mixtures of the first compound and second compound. As shown in the graphs and table, except for the combination of glutathione and TCEP, detection of the minimum bacterial concentration was possible in less than 10 minutes, similar to addition of the first compound or second compound alone. This indicated a result that detection is possible in less than 10 minutes by room temperature analysis and 50° C. analysis at equivalent sensitivity even when the first compound and second compound are used in admixture, similar to their use alone.
(A) Mixed Preparations of First Compounds and Second Compounds
The first compounds and the second compounds were mixed in combinations shown in Table 3. Mixing concentrations were adjusted by dissolving 55.5 mM of each of a first compound and a second compound in 50 mM tris-maleate buffer solution pH 8.5 and thereafter mixing equivalent amounts of the first compound and the second compound such that the concentration of each would be 27.75 mM.
(B) Dry Retention of Substrate
The prepared substrate was mixed with 50 mM tris-maleate buffer solution pH 8.5 such that a volume ratio would be 9:1 and 10 mm paper disks (obtained from Advantec Co., Ltd.) were impregnated with 80 μL of the mixture per disk. The paper disks were then dried overnight at 25° C.
(C) Preparation of P.g
The bacterial culture solution after 24 hours of culturing was 10-fold serially diluted with the prepared mixtures of first compounds and second compounds to prepare bacterial suspensions of 1.0×106 cfu/mL, 1.0×105 cfu/mL, 1.0×104 cfu/mL, and 1.0×103 cfu/mL.
(D) Test Method
Two of the paper disks, impregnated with the substrate and then each impregnated with 80 μL of the prepared P. g bacterial suspension, were prepared and left to stand respectively for 2 minutes, 5 minutes, 8 minutes, 10 minutes, 15 minutes, and 20 minutes, one at room temperature and the other at 50° C. The former shall be referred to as room temperature analysis and the latter as 50° C. analysis. Thereafter, 30 μL of the coloring solution was dripped onto each paper disk and coloration of each paper disk was observed and judged with the naked eye.
(E) Results
It has become clear that while sufficient sensitivity cannot be obtained in room temperature analysis for enzyme activity having an optimal temperature of 50 to 60° C. and the analysis time is also lengthened, the present invention enables, by dry retention in a water absorbent material or addition to an sample of a first compound having an antioxidant effect or a compound having action of protecting SH groups and cleaving disulfide bonds, analyzing the enzyme activity in less than 10 minutes at a sensitivity equivalent to 50° C. analysis even with room temperature analysis.
The present invention is useful for technologies in periodontopathic bacteria detection and diagnosis for easily performing ascertainment to make risk judgments and checks of degree of progress of periodontal disease as well as checks of treatment effects, etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2019-083482 | Apr 2019 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/016256 | 4/13/2020 | WO | 00 |
