JP4792585B2 - Rapid detection method for oral bacteria - Google Patents

Description

  The present invention relates to a method for detecting oral bacteria, particularly oral infection-related bacteria, and a kit for diagnosis of bad breath and periodontal disease.

  In recent years, dental caries and periodontal disease, which are two major diseases of dentistry, are considered to be infections caused by oral infection-related bacteria. Until now, bacterial tests, which are basic tests for infectious diseases, have not been performed for these diseases, but they should be performed as long as the diseases are bacterial infections. In addition, bacterial tests play an extremely important role in the determination and evaluation of therapeutic effects and the determination of disease prognosis. One of the reasons why bacterial testing has not been actively introduced in dentistry is that there was no simple and accurate method for testing bacteria. In medical practice, the bacterial culture method is only recognized as a simple bacteria test for endodontic treatment, but it is difficult to say that it is popular in general dental clinics. This is thought to be due to problems in the operability and rapidity of the bacterial culture test.

  In the dental field, periodontal disease, bad breath, and pulpitis, a sequelae of dental caries, are the main reasons patients visit hospitals, and the number of patients is very large. Moreover, interest in halitosis has increased due to the improvement of hygiene awareness, and the number of dental patients who have chief complaints of halitosis has been increasing in recent years. According to the “Health and Welfare Trend Survey 1999” by the Ministry of Health and Welfare, 15.8% of men, 13.2% of women, and 14.5% of people have bad breath. It can be said that oral health professionals are entering an era in which they should actively address bad breath. These diseases are commonly infectious diseases caused by oral bacteria. Therefore, a simple and inexpensive method for detecting the bacterial group is indispensable for diagnosing and treating diseases, and its needs are high.

  Since the ratio of periodontal disease patients has bad breath has been high, the relationship between periodontal disease and bad breath has been pointed out. Miyazaki et al. Examined 2,672 subjects aged 18 to 64 years, examining the health and cleaning status of teeth and periodontal tissue, and at the same time, using portable sulfide monitors for volatile sulfides contained in exhaled breath. It is measured using. As a result, it has been clarified that the amount of volatile sulfide in breath correlates with the periodontal tissue health and the amount of tongue coating (Non-patent Document 1). In addition, the group with deep periodontal pockets (probing depth, 4 mm or more) and the group with bleeding during probing clearly show that high concentration of volatile sulfide is detected from the gingival crevice compared to the group without (Non-Patent Document 2). Based on the above, several reports have been made on the ability of periodontopathic bacteria to produce halitosis-causing substances. Among them, methyl mercaptan, which is prominently detected in the expiration of periodontal disease patients, is Porphyromonas gingivalis ( It has been reported that homocysteine-degrading enzymes of obligate anaerobes such as Porphyromonas gingivalis, Fusobacterium nucleatum, and Treponema denticola are substances resulting from the degradation of methionine (non-patented) Literature 3-5). It has been reported that these bacteria exhibit remarkable cysteine resolution in addition to homocysteine resolution.

  On the other hand, the presence of bacteria having remarkable cysteine resolution has been reported in bacteria other than periodontal pathogenic bacteria. Yoshida et al. Have revealed that the gram-positive bacterium Streptococcus anginosus exhibits high cysteine resolution (Non-patent Document 6). The cysteine-degrading enzyme of this bacterium modifies hemoglobin contained in erythrocytes to cause hemolysis (Non-patent Document 7), and the bacterium is detected frequently from the abscess site. Has attracted attention as a causative fungus

  Diagnosis of oral infection requires effective combination of clinical symptoms, diagnostic imaging, and bacteriological examination, but the bacterial culture method that actually isolates and identifies bacteria is not easy to collect specimens by site, Depending on the type of bacteria, there is a limit because the detection rate is not necessarily high and the culture takes some time. In order to eliminate these disadvantages, a genetic test method for oral infection-related bacteria has been developed, and many studies have been reported on detection methods using the polymerase chain reaction (PCR) method. However, it requires expensive equipment such as a thermal cycler, a separate detection process, lack of simplicity that can be performed on the chair side, and high cost per test. Disadvantages remain unresolved. Therefore, the Loop-mediated Isothermal Amplification (LAMP) method has been developed in recent years as a simpler, faster and more accurate genetic testing method than the PCR method, but the cost improvement is insufficient and the detection sensitivity is extremely high. There is a problem that it is difficult to distinguish between a lesion site in which a small amount of bacterial DNA remains and a site in which bacteria are actively proliferating. For the examination of bad breath, sensory tests and gas chromatography methods are mainly used. The sensory test determines the degree of odor by a tester, has poor quantitativeness, and the patient's satisfaction with the evaluation of the results is inferior to that using an apparatus or a test reagent. On the other hand, gas chromatography directly quantifies substances that cause bad breath, and is highly reliable, but has high equipment and maintenance costs, and is not suitable for general clinics.

As a test reagent using the enzyme activity of bacteria as an index, “Periocheck (registered trademark)” of Sunstar Co., Ltd. has already been commercialized. This is to determine the presence of bacteria by detecting peptidase activity such as Porphyromonas gingivalis (formerly Bacteroides gingivalis) and Treponema denticola, which are periodontal pathogenic bacteria (Patent Document 1). However, general practitioners such as some periodontopathic bacteria that do not have the same peptidase activity, that the expiry date is short because the detection reagent contains another enzyme, and that the reagent is expensive. However, there are still many problems to use for inspection purposes.
JP-A-1-144997 Miyazaki, H et al., `` J. Periodontol '' 1995, 66, 679-684. Coil, JM et al., `` J. Clin. Dent. '', 1992, volume 3, 97-103. Yoshimura, M. et al., “Infect. Immun.”, 2000, Vol. 68, 6912-6916. Yoshimura, M. et al., `` FEBS Lett. '', 2002, 523, 119-122. Fukamachi, H. et al., “Biochem. Biophys. Res. Commun.” 2005, 331, 127-131. Yoshida, Y. et al., "Biochem. Biophys. Res. Commun.", 2002, 300, 55-60. Yoshida, Y. et al., `` Microbiology. '', 2002, 148, 3961-3970.

  An object of the present invention is to provide a rapid detection system for oral bacteria, preferably oral infection-related bacteria, which can be rapidly, specifically, inexpensively and easily used in clinical practice.

  In view of the above problems, the present inventors have focused on homocysteine-degrading enzymes and cysteine-degrading enzymes that are frequently found in oral bacteria, particularly oral infection-related bacteria, and have completed the present invention.

That is, the present invention is as follows.
(1) A method for detecting oral bacteria, comprising measuring homocysteine and / or cysteine-degrading enzyme activities in a specimen.
(2) The detection method according to (1), wherein the oral bacteria are periodontal pathogenic bacteria, halitosis-causing substance-producing bacteria, root canal infection bacteria, or apical foci bacteria.
(3) The detection method according to (1) or (2), wherein the measurement of the enzyme activity is a measurement of coloration generated by a reaction between hydrogen sulfide and bismuth.
(4) A method for identifying periodontopathic bacteria characterized by measuring homocysteine-degrading enzyme activity in a sample collected from the oral cavity.
(5) A method for identifying a pathogenic bacterium for pulpitis, apical periodontitis or root canal infection, characterized by measuring cysteine-degrading enzyme activity in a sample collected from the oral cavity.
(6) The identification method according to (4) or (5), wherein the measurement of the enzyme activity is a measurement of coloration generated by a reaction between hydrogen sulfide and bismuth.
(7) collecting a sample from the oral cavity;
Adding the collected sample to a reaction solution containing bismuth and homocysteine or cysteine and keeping the temperature warm;
A method for detecting oral bacteria, comprising a step of measuring the coloration of the reaction solution after the heat retaining step and a step of identifying a group of oral bacteria present in the oral cavity based on the measurement result.
(8) The detection method according to (7), wherein the oral bacteria group is composed of a high homocysteine decomposition activity group, a medium homocysteine decomposition activity group, a high cysteine decomposition activity group, and a medium cysteine decomposition activity group.
(9) The detection method according to (7), wherein the high to medium homocysteine degrading activity group is Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Treponema dendicola, and Tanerella faucisia.
(10) The detection method according to (7), wherein the high to medium cysteine-degrading activity group is Streptococcus anginasus, Prevotella nigres sens, and Enterococcus faecalis.
(11) The detection method according to any one of (1) to (3) and (7) to (10), which is used as an index for treatment of bad breath, periodontal disease, pulpitis or endodontic infection. The identification method according to any one of 4) to (6).
(12) A reagent for detecting oral bacteria by measuring the activity of homocysteine or cysteine-degrading enzyme in a specimen, the reagent containing bismuth and homocysteine or cysteine.
(13) The reagent according to (12), further comprising a surfactant.
(14) A diagnostic kit for halitosis, periodontal disease, pulpitis or root canal infection, comprising a collection means for collecting a sample from the oral cavity and the reagent according to (12) or (13).

  The method for detecting oral bacteria according to the present invention is performed by heating a collected specimen or sample in a reaction solution for a certain period of time. Since it is possible by measurement, it is much simpler and quicker than conventional methods. In addition, the method for identifying a pathogenic bacterium of periodontal disease, pulpitis or root canal infection according to the present invention can similarly determine the presence or absence of a pathogenic bacterium simply and quickly. In addition, the method for detecting periodontal disease, pulpitis, root canal infection or bad breath according to the present invention can similarly detect the presence or absence of the disease in a simple and rapid manner. Furthermore, the reagents necessary for the detection method and identification method of the present invention are very inexpensive and have a very high application value as a widely used detection agent. Therefore, the detection method, identification method, reagent and kit of the present invention provide a quick, simple, inexpensive and accurate diagnosis for patients suffering from oral infections such as periodontal disease, pulpitis, root canal infection and bad breath. It is very useful for early detection and treatment of the infectious disease.

FIG. 1 is a diagram in which oral bacteria are arranged in a group having a high enzyme activity, a middle group, and a group having a low level, with the respective homocysteine and cysteine-degrading enzyme activities as coordinates. FIG. 2 is a graph showing the correlation between the detection result of enzyme activity using a bismuth reaction solution and the number of bacteria in the reaction solution. FIG. 3 shows the change with time of the absorbance of the reaction solution with respect to the reaction time. FIG. 4 shows the results of measuring the homocysteine and cysteine-degrading enzyme activities of each oral bacterium after 30 minutes and 60 minutes, respectively. FIG. 5 shows the results of measuring the homocysteine and cysteine-degrading enzyme activities of each oral bacterium after 30 minutes and 60 minutes, respectively. FIG. 6 shows the results of measuring the homocysteine and cysteine-degrading enzyme activities of each oral bacterium after 30 minutes and 60 minutes, respectively. FIG. 7 shows the results of measuring the homocysteine and cysteine-degrading enzyme activities of each oral bacterium after 30 minutes and 60 minutes, respectively. FIG. 8 shows the results of measuring the homocysteine and cysteine-degrading enzyme activities of each oral bacterium after 30 minutes and 60 minutes, respectively. FIG. 9 is a graph showing the absorbance at 405 nm of the reaction solution with respect to each temperature condition over time. The horizontal axis represents temperature, and the vertical axis represents absorbance. FIG. 10 is a graph showing the absorbance at 405 nm of the reaction solution with respect to each pH condition over time. The horizontal axis represents pH, and the vertical axis represents absorbance. FIG. 11 is a graph showing the absorbance at 405 nm of the reaction solution over time when Triton X-100 of each concentration was added. The horizontal axis represents the concentration of Triton X-100, and the vertical axis represents the absorbance. FIG. 12 is a graph showing the reproducibility of a sample collected using paper points. The horizontal axis represents pH, and the vertical axis represents absorbance. FIG. 13 is a graph showing the absorbance at 405 nm of the reaction solution over time when sodium hydroxide is added so as to have final concentrations of 0.1, 0.15, and 0.2N. FIG. 14 is a graph showing the effect of human-derived tissue on the detection method of the present invention in Reference Example 1. FIG. 15 is a graph showing the correlation between the collected periodontal pocket depth and absorbance for the homocysteine-degrading enzyme activity of a sample collected using paper points. The horizontal axis indicates the depth of the periodontal pocket, and the vertical axis indicates the absorbance. FIG. 16 is a graph showing the correlation between the collected periodontal pocket depth and absorbance of the collected cysteine-degrading enzyme activity of the sample collected using paper points. The horizontal axis indicates the depth of the periodontal pocket, and the vertical axis indicates the absorbance.

(Method for detecting oral bacteria)
The method for detecting an oral bacterium of the present invention (hereinafter also referred to as the detection method of the present invention) is a method for measuring oral cysteine or cysteine-degrading enzyme activity in a sample, thereby measuring oral bacteria contained in the sample, preferably oral infections. It detects related bacteria.

Homocysteine and cysteine are decomposed by homocysteine-degrading enzyme and cysteine-degrading enzyme, respectively, to produce hydrogen sulfide.
Homocysteine-degrading enzyme is an enzyme generally known as L-methionine-α-deamino-γ-mercaptomethane-lyase and is known to produce methyl mercaptan using methionine as a substrate. It has been reported to degrade cysteine well. The reaction formula is as follows.
L-homocysteine → hydrogen sulfide + α-ketobutyric acid + ammonia L-methionine → methyl mercaptan + α-ketobutyric acid + ammonia In addition, cysteine-degrading enzyme is an enzyme known as βC-S lyase, and its reaction formula is as follows: is there.
L-cysteine → hydrogen sulfide + pyruvic acid + ammonia

The detection method of the present invention is carried out by measuring the activity of homocysteine and / or cysteine-degrading enzyme in a specimen by measuring hydrogen sulfide produced by the enzyme reaction, utilizing the above-mentioned enzymatic properties. The method for measuring the generated hydrogen sulfide is not particularly limited, and a known method can be adopted.
In a preferred embodiment, the method for measuring hydrogen sulfide includes a modification of the method of Claesson et al. (See Claesson, R. et al., Oral Microbiol. Immunol. 1990, Vol. 5, 137-142). Specifically, first, a sample (sample collected from the oral cavity) is added to the bismuth reaction solution. When hydrogen sulfide is generated by the enzyme reaction, bismuth reacts with hydrogen sulfide and turns into black bismuth sulfide. After heating for a certain time, the target oral bacteria, preferably oral infection-related bacteria, can be detected by observing the blackening of the reaction solution or measuring the absorbance at 405 nm.
The detection method of the present invention may be performed by measuring methyl mercaptan produced using methionine as a substrate. As a method for detecting methyl mercaptan, a gas chromatograph can be used as a direct detection method. Further, as a method for indirectly detecting, by using a reagent containing 3-methyl-2-benzothiazolinone hydrazone, the production of α-ketobutyric acid, which is a byproduct of the methyl mercaptan formation reaction, is measured by measuring the absorbance at 335 nm. There is a way to detect.

(Oral bacteria)
In the present invention, oral bacteria refer to bacteria that are widely present in the oral cavity. Oral infection-related bacteria refer to bacteria that cause oral infections among pathogenic bacteria (pathogenic bacteria). For example, periodontal pathogenic bacteria, bacteria producing halitosis, root canal infections Examples include bacteria and apical foci bacteria. Specific examples of oral bacteria include Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Prevotella denticola, Prevotella melaninogenica Prevotella melaninogenica Prevotella denticola melaninogenica, Prevotella pallens, Prevotella loescheii, Prevotella veroralis, Prevotella bivia, Prevotella nigrescens, str ococcus Streptococcus , Streptococcus milleri, Streptococcus anginosus, Streptococcus mutans (Strep tococcus mutans), Streptococcus sobrinus, Streptococcus salivarius, Streptococus gordonii, Enterococcus faecalis, etc.

  These oral bacteria are shown in FIG. 1 as a table divided into a group having high homocysteine and cysteine-degrading enzyme activities, an intermediate group, and a low group. Porphyromonas gingivalis and Fusobacterium nucleatum that showed high homocysteine-degrading enzyme activity and high cysteine-degrading enzyme activity are bacteria known as periodontopathic bacteria. It has also been reported that hydrogen sulfide produced by homocysteine and cysteine-degrading enzymes of these bacteria and methyl mercaptan produced from methionine by the enzyme are correlated with the intensity of bad breath (Persson, S. et al., Oral Microbiol. Immunol. 1990, Vol. 5, 195-201). Similarly, Prevotella intermedia with moderate homocysteine-degrading enzyme activity and Prevotella nigressense with moderate cysteine-degrading enzyme activity are also frequently detected in the pockets of periodontal patients. ing. On the other hand, Streptococcus anginasus which showed high cysteine-degrading enzyme activity is a bacterium frequently detected from the site of abscess (Whitworth, JM et al., J. Med. Microbiol. 1990, 33, 135-151), It has been implicated in abscess formation. In addition, Enterococcus faecalis that showed moderate cysteine-degrading enzyme activity has been reported to be detected in the root canal of approximately 40% of teeth with apical lesions (Pinheiro, ET et al., Oral Microbiol). Immunol. 2003, Vol. 18, 100-103). There is a report that pulpitis is caused by complex infections such as Streptococcus anginasus, Fusobacterium nucleatum, Porphyromonas gingivalis and Treponema denticola (Katsuaki Okuda, latest oral microbiology, Issei Publishing, 2005, 402-404). reference). The present invention can also easily classify oral bacteria into the above-mentioned groups by combining measurement of homocysteine-degrading activity and cysteine-degrading activity.

(Method of identifying pathogenic bacteria of periodontal disease, pulpitis or root canal infection)
The present invention provides a method for identifying periodontal disease pathogenic bacteria by measuring homocysteine-degrading enzyme activity in a sample collected from the oral cavity in the same manner as in the detection method described above.
Further, according to the present invention, the cysteine-degrading enzyme activity in a sample collected from the oral cavity is measured in the same manner as in the above detection method, and pathogenic bacteria of pulpitis, apical periodontitis or root canal infection are detected. A method of identifying can be provided.

Existing methods can be used to collect samples from the oral cavity. For example, the gingival crevicular fluid and saliva in the periodontal pocket can be obtained using commercially available paper points (stiff sticks) and filter paper. Also, plaque can be collected using a cotton swab or curette. Furthermore, when collecting tongue coating from the tongue surface, a filter paper, a spatula, or the like may be used.
The collected sample may be used for the detection method of the present invention as it is, or after culturing as described in the Examples, the culture solution (bacterial solution) may be used for the detection method of the present invention. The former is preferable for shortening the time.

Hereinafter, the hydrogen sulfide detection system will be described in detail.
(Reaction solution)
In the present invention, the reaction solution basically contains bismuth and homocysteine or cysteine.
As the bismuth, a compound that can react with hydrogen sulfide in a solution can be used without limitation, and examples thereof include bismuth chloride, bismuth acetate, bismuth phosphate, and bismuth fluoride.
For homocysteine, it is desirable to use the L form, but the DL form may also be used. As for cysteine, it is desirable to use L-form, but DL-form may also be used.
The reaction solution may contain a buffer, and the buffer is not particularly limited as long as it is usually used. For example, a triethanolamine buffer (eg, triethanolamine hydrochloride), trishydrochloric acid Examples thereof include a buffer solution and a phosphate buffer solution.
The reaction solution preferably further contains a surfactant in order to dissolve the bacterial cell membrane and release the enzyme outside the cells. Examples of the surfactant include Triton X-100, Nonidet P40, Tween 20, and the like, and Triton X-100 is preferably used.
Ethylenediaminetetraacetic acid (EDTA) or the like is used for pH adjustment of the reaction solution.
Two kinds of reaction solutions are prepared by containing either homocysteine or cysteine in the reaction solution. A reaction solution is selected according to each decomposing enzyme activity of oral bacteria.
Preferably, it is in the range of pH 8 to pH 9.5 for the reaction solution for detecting homocysteine-degrading enzyme activity, and in the range of pH 8.0 to pH 9.0 for the reaction solution for detecting cysteine-degrading enzyme activity.

  In the reaction solution, the final concentration of bismuth is usually 0.1 to 5.0 mM, preferably 0.5 to 2.0 mM, and the final concentration of homocysteine or cysteine is usually 10 to 40 mM, preferably 15 to 25 mM. It is. Below these concentrations, the reaction does not occur sufficiently, and above that, background increases. Within these concentrations, the reaction between the enzyme and substrate of the bacteria in the sample and the color reaction of hydrogen sulfide and bismuth, which are reaction products, proceed quantitatively. When the reaction solution contains a surfactant, the concentration is usually 0.1 to 2.0%, preferably 0.2 to 0.5%.

(Heat retention process)
Next, the optimum conditions for adding a sample to the reaction solution will be described.
The contact between the sample and the reaction solution is performed, for example, in a test tube, a microtube, a cell, a vial, or the like.
When adding a specimen culture solution (bacterial solution) to the reaction solution, it is preferable to suspend the sample so that the number of bacteria is about 0.3 to 2.5 × 10 ⁸ per 500 μl of the reaction solution.
Moreover, when the paper point which collect | recovered the sample is immersed in a reaction liquid directly and made to contact, the reaction liquid used by one measurement is 0.5-1 ml normally.
When looking at the homocysteine-degrading enzyme activity, a relatively high temperature condition in the range of 35 ° C. to 50 ° C. is suitable, and the reaction is usually allowed to proceed for 30 minutes or longer, preferably about 60 minutes. On the other hand, when looking at cysteine-degrading enzyme activity, about 30 ° C. to 40 ° C. is suitable, and the reaction is usually allowed to proceed for 30 minutes or longer, preferably about 60 minutes.

(Measurement process)
In the detection method of the present invention, the reaction is allowed to proceed for 30 minutes or longer, preferably about 60 minutes under the above conditions, and then the absorbance of the reaction solution is measured, or the reaction solution is visually observed for blackening. Measure the color of and identify the presence of oral bacteria and periodontal pathogens.
When measuring the light absorbency of a reaction liquid, the measuring method is not specifically limited, It measures using the existing methods, such as a spectrophotometer. Specifically, for example, after the reaction has sufficiently progressed, the absorbance of the reaction solution at 405 nm is measured using a spectrophotometer.
In addition, when measuring the absorbance of the reaction solution, the absorbance of the reaction solution increases over time in the same state. Therefore, assuming that measurement could not be performed immediately after completion of the reaction, the reaction was stopped and the absorbance was measured for a certain period of time. Is preferably maintained in a stable state. In this case, sodium hydroxide is added to stop the reaction. The concentration of sodium hydroxide to be added is 0.1 to 0.3N as a final concentration, preferably 0.15 to 0.2N.
In addition, when observing the blackening of the reaction solution with the naked eye, the degree of blackening of the reaction solution can be determined by, for example, a standard color chart or other color sheet issued by the Japanese Standards Association in accordance with the Japanese Industrial Standard (JIS Z 8721). You may determine by comparing.

  As a determination method, if the reaction solution containing homocysteine is colored, it is assumed that the infection is an obligate anaerobic bacterium that is a periodontopathic bacterium. Moreover, if the reaction liquid containing cysteine is colored, it is determined that there is a bacterial infection that causes pulpitis, apical periodontitis, and endodontic infection. Although the degree of bad breath can be objectively evaluated by the degree of coloration of the reaction liquid containing homocysteine and cysteine, the bad breath is more severe when the degree of coloration of the reaction liquid containing homocysteine is strong. It is determined.

  Thus, by measuring the homocysteine and cysteine-degrading enzyme activities in the specimen, it is possible to easily diagnose the presence or absence of periodontal disease, pulpitis, apical periodontitis, and endodontic infection.

(Detection reagent)
Furthermore, the present invention provides a reagent for detecting oral bacteria, preferably oral infection-related bacteria, by measuring homocysteine or cysteine-degrading enzyme activity in a specimen. The reagent of the present invention contains bismuth and homocysteine or cysteine. Further, the reagent of the present invention may contain a surfactant.
The reagent of the present invention is preferably stored in the form of a solid that is dry powdered or granulated by a known method with respect to homocysteine and cysteine as substrates, and is prepared and used in a buffer solution (solvent) at the time of use. desirable.

(kit)
Furthermore, the present invention provides a kit for diagnosing bad breath, periodontal disease, pulpitis or root canal infection, which includes a collecting means for collecting each sample from the oral cavity and the detection reagent. Here, as described above, the affected part includes a periodontal pocket, tongue surface, tooth surface, etc., examples of the specimen include gingival crevicular fluid, saliva, plaque, tongue coating, etc. Examples include filter paper, cotton swabs, and curettes. The present invention also provides a therapeutic effect determination kit for root canal treatment by detecting the presence or absence of oral bacteria, which further comprises a paper point for collecting bacterial cells from the root canal and the detection reagent. To do. These kits may include a color sheet used for observing the blackening of the reagent.

(Use)
Since the detection method and kit of the present invention can detect various oral bacteria, particularly oral infection-related bacteria such as periodontal pathogenic bacteria, the detection method and kit of the present invention are oral infections, Periodontitis (chronic periodontitis, necrotizing ulcerative periodontitis, gestational periodontitis, apical periodontitis, etc.), pulpitis (acute purulent pulpitis, chronic ulcerative pulpitis) It is very useful for diagnosis and prevention of root canal infection. Further, the detection method and kit of the present invention are used as an index for the treatment of the above-mentioned diseases, and deal with problems related to various diseases in the oral cavity, such as determination of therapeutic effects in root canal treatment and measurement of the degree of bad breath. It is also possible.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples. However, the examples are described for explaining the present invention, and do not limit the present invention.
Each strain used in the examples of the present invention is shown in Table 1.

  The bismuth reaction solution used in the examples of the present invention is 200 mM triethanolamine hydrochloride containing 0.5 mM bismuth chloride, 10 mM EDTA, and 20 mM DL-homocysteine or 20 mM L-cysteine.

Example 1: Correlation between detection result of enzyme activity and number of bacteria in reaction solution In order to confirm that the detection result of enzyme activity using bismuth reaction solution correlates with the number of bacteria in reaction solution, porphyro The bacterial solution of Monas gingivalis (ATCC33277 strain) was serially diluted and 10 × 10 ⁸ , 5 × 10 ⁸ , 2.5 × 10 ⁸ , 1.25 × 10 ⁸ , 0.63 × 10 ⁸ , 0.31 × The suspension was suspended in a bismuth reaction solution (pH 8.0) containing homocysteine to 10 ⁸ , 0.16 × 10 ⁸ and 0.07 × 10 ⁸ CFU / ml, heated at 37 ° C. for 60 minutes, and then at 405 nm. Absorbance was measured.
As a result, as shown in FIG. 2, it was revealed that the absorbance increased in correlation with the number of bacteria in the reaction solution. It was also found that the increase in absorbance correlates with the reaction time as shown in FIG.

Example 2 Measurement of Homocysteine and Cysteine Degrading Enzyme Activities of Each Oral Bacteria The homocysteine and cysteine degrading enzyme activities of each oral bacterium were compared. Each bacterium was anaerobically overnight at 37 ° C using a medium containing 5 g Yeast Extract, 1 g L-cysteine hydrochloride, 5 mg hemin and 1 mg vitamin K (enriched BHI broth) per liter of Brain Heart Infusion. After culturing, the bacterial solution that reached the stationary phase was transferred to a fresh medium and cultured until the logarithmic growth phase was reached. Thereafter, the culture solution was centrifuged to remove the supernatant, and the absorbance at 600 nm was adjusted to 1.0 using a fresh medium. After centrifuging 500 μl of the bacterial solution thus obtained and removing the supernatant, the bacterial cells are suspended in 500 μl of a bismuth reaction solution (pH 8.0) containing 1% Triton X-100 and incubated at 37 ° C. for 30 minutes or 60 minutes. After the reaction, the absorbance at 405 nm was measured.
The results are shown in FIGS. This result revealed that the detection of homocysteine and cysteine-degrading enzyme activities is useful as an index to know the presence of oral bacteria.

Example 3: Examination of reaction conditions (1) Reaction temperature After culturing Porphyromonas gingivalis (ATCC33277 strain) anaerobically overnight at 37 ° C using enriched BHI broth, the bacterial solution that reached the stationary phase was freshly prepared. The medium was subcultured and cultured until the logarithmic growth phase was reached. Thereafter, the culture solution was centrifuged to remove the supernatant, and the absorbance at 600 nm was adjusted to 1.0 using a fresh medium. After centrifuging 500 μl of the above bacterial solution and removing the supernatant, the bacterial cells were suspended in 500 μl of a bismuth reaction solution (pH 8.0). Thereafter, the absorbance at 405 nm was observed over time while heating under changing conditions in the range of 25 ° C. to 50 ° C.
The results are shown in FIG. 9 (in the figure, the vertical axis represents absorbance). From this, it was found that the reaction under a relatively high temperature condition in which the reaction temperature is in the range of 35 ° C. to 50 ° C. is suitable for the homocysteine degrading enzyme activity. On the other hand, it was found that the cysteine-degrading enzyme activity is suitably about 30 to 40 ° C.
(2) Reaction pH
After centrifuging 500 μl of the bacterial solution prepared by the method described in (1) and removing the supernatant, the cells are suspended in 500 μl of a bismuth reaction solution of pH 7.0 to pH 9.5 and heated at 37 ° C. The absorbance at 405 nm was observed over time.
The results are shown in FIG. 10 (in the figure, the vertical axis represents the absorbance). From this, it was revealed that the homocysteine degrading enzyme activity is suitable for the reaction in the range of pH 8 to pH 9.5. On the other hand, the pH of the reaction solution suitable for detection of cysteine-degrading enzyme activity was pH 8.0 to pH 9.0.
(3) Surfactant Since homocysteine-degrading enzyme is an intracellular enzyme, it is considered that detection efficiency is better if the bacteria are lysed by some method so that the intracellular enzyme can directly contact the substrate. Therefore, it was decided to compare the increase in absorbance between the case where a surfactant or the like was added to a reaction solution containing an equal amount of bacterial cells and the case where a surfactant was not added. After centrifuging 500 μl of the bacterial solution prepared by the method described in (1) and removing the supernatant, 500 μl of a bismuth reaction solution containing 0.1% to 2% Triton X-100 or not containing bismuth reaction solution ( The suspension was suspended at pH 8.0), heated at 37 ° C., and the absorbance at 405 nm was observed over time.
The results are shown in FIG. 11 (in the figure, the vertical axis represents absorbance, and the horizontal axis represents Triton X-100 concentration). It was revealed that when Triton X-100 was added to the reaction solution, the detection efficiency was improved as compared with the case where Triton X-100 was not added.

Example 4: Confirmation of reproducibility of sample collected using paper points When actually collecting a sample from a patient, it is performed using a collecting means such as a paper point. Therefore, the following experiment was conducted to confirm that the same results as when the cells were directly suspended in the reaction solution were obtained even when the cells attached to the paper point were immersed in the reaction solution. First, 10 ml of the bacterial solution adjusted so that the absorbance at 600 nm was 1.0 was centrifuged, and the cells were collected and suspended in 500 μl of fresh medium. The paper point was immersed in this bacterial solution for 10 seconds and immediately immersed in 500 μl of a bismuth reaction solution containing 0.5% Triton X-100 from pH 7.0 to pH 9.5. Thereafter, the absorbance at 405 nm was observed over time while heating at 37 ° C.
The results are shown in FIG. 12 (in the figure, the vertical axis represents the absorbance). From this result, it was confirmed that the enzyme activity could be detected by a method in which bacteria were adsorbed on a paper point and then immersed in a bismuth reaction solution. It was also revealed that the detection efficiency of a sample collected using a paper point can be improved nearly twice by adding Triton X-100 to the reaction solution.

Example 5: Examination of reaction stop conditions After centrifuging 500 μl of the bacterial solution prepared by the method described in Example 3 (1) and removing the supernatant, 500 μl of bismuth reaction solution (pH 8.0) was obtained. ) And warmed at 37 ° C. for 20 minutes. Thereafter, sodium hydroxide was added so that the final concentrations were 0.1, 0.15, and 0.2 N, and the absorbance at 405 nm was observed over time at room temperature.
The results are shown in FIG. According to FIG. 13, the reaction was not stopped at the final concentration of 0.1N, and although the value was small at 0.15N, a moderate increase in absorbance was observed. In contrast, when sodium hydroxide having a final concentration of 0.2 N was added, stable measurement values could be obtained over 3 days.

Reference Example 1: Examination of the effects of human-derived tissue In sites where samples from patients are actually collected, such as periodontal pockets, the presence of bleeding due to inflammation or detached epithelial cells is often observed. Periodontal pockets of patients suffering from periodontal disease are easy to bleed and are likely to be contaminated with blood when samples are collected. Therefore, in order to investigate whether human-derived cells affect the detection of enzyme activity, the peripheral blood and human gingival fibroblast-derived cell lines were added to the reaction solution, and the absorbance was compared with the case without addition. It was.
First, Porphyromonas gingivalis (ATCC33277 strain) was anaerobically cultured overnight at 37 ° C. using enriched BHI broth, and the bacterial solution reaching the stationary phase was transferred to a fresh medium until the logarithmic growth phase was reached. Cultured. Thereafter, the culture solution was centrifuged to remove the supernatant, and the absorbance at 600 nm was adjusted to 1.0 using a fresh medium. The supernatant was removed by centrifuging 500 μl of the above bacterial solution. The microbial cells thus obtained were added to 0.5 or 1 μl of human peripheral blood or 1 × 10 ⁴ HSC-2, which is a human gingival fibroblast-derived cell line (1% Triton X- (Including 100). Thereafter, the mixture was heated at 37 ° C. for 30 minutes, and the absorbance at 405 nm was measured. Peripheral blood was collected from healthy adults in the presence of heparin, and HSC-2 was cultured in RPMI 1640 medium containing 10% bovine serum, 2 mM L-glutamine, 100 μg / ml streptomycin and 6 μg / ml penicillin. Using.
The results are shown in FIG. As a result, in the case where 1/500 amount of blood was contained in the reaction solution, the numerical value was raised overall due to the color of the blood itself, but 1/1000 amount of blood and 1 × 10 ⁴ It was found that the detection of homocysteine and cysteine-degrading enzyme activities using bismuth sulfide production as an index is not affected in comparison with the values when blood and cells are not mixed.

Example 6: Examination of correlation between depth of periodontal pocket and absorbance of bismuth reaction In order to examine the relationship between this detection system and the pathology of periodontal disease, gingival crevicular fluid was collected, The relationship between homocysteine and cysteine-degrading enzyme activities was examined. The method for collecting human gingival crevicular fluid was as follows. First, the oral cavity was divided into 6 fractions, and the deepest pocket of each fraction was used as a sample collection target. Next, after removing the surrounding saliva from the periodontal pocket to be sampled, insert a paper point and let stand for 10 seconds, put it in a bismuth reaction solution containing 0.5% TritonX-100, and decompose homocysteine The enzyme activity was detected at 45 ° C. for 60 minutes, and the cysteine-degrading enzyme activity was detected at 37 ° C. for 60 minutes. The absorbance at 405 nm was measured.

  15 and 16 show the analysis results of 69 outpatients of 12 patients from Kyushu Dental University Hospital. FIG. 15 shows the analysis results for homocysteine decomposition activity and periodontal pocket depth, and FIG. 16 shows the analysis results for cysteine decomposition activity and periodontal pocket depth. It was revealed that the enzyme activity was higher as the pocket depth was deeper for any enzyme activity. In particular, it has been clarified that the tendency is strong for the homocysteine decomposition activity.

  ADVANTAGE OF THE INVENTION According to this invention, the rapid detection system of oral bacteria which can be used practically at a dental clinical site is provided, and oral infection related bacteria can be detected quickly, specifically, inexpensively and easily.

  This application is based on Japanese Patent Application No. 2005-318312 (filing date: November 1, 2005) filed in Japan, the contents of which are incorporated in full herein.

Claims (8)

Buccal or we adopted collected samples, the step of incubating in addition to the reaction the reaction solution and containing bismuth and cysteine containing bismuth and homocysteine,
A step of measuring the coloration of the reaction solution after the heat retaining step, and a step of identifying a group of oral bacteria present in the oral cavity based on the measurement result ,
including,
A method for detecting oral bacteria , comprising measuring homocysteine and cysteine-degrading enzyme activities in a specimen . The oral bacteria group has a high homocysteine-decomposing activity and a high cysteine-degrading activity, a group having a moderate homocysteine-degrading activity and a low cysteine-degrading activity, a group having a low homocysteine-degrading activity and a high cysteine-degrading activity, and a low The detection method according to claim 1 , wherein the detection method comprises a group having homocysteine degrading activity and moderate cysteine degrading activity . The high group with a homocysteine degradation activity and a high-cysteine decomposition activity Gapo a Rufiromonasu gingivalis and Fusobacterium nucleatum, a group having a homocysteine degradation activity and low-cysteine decomposition activity of the moderate a Prevotella intermedia, the low homocysteine degradation activity and a group having a high cysteine decomposition activity is Streptococcus anginosus, claim 2 group with low homocysteine degradation activity and moderate-cysteine decomposition activity is Prevotella Niguresusensu and Enterococcus faecalis The detection method according to. The detection method according to any one of claims 1 to 3, wherein the reaction solution further contains a surfactant. Halitosis, periodontal disease, detection how according to any one of claims 1-4 for use as an indicator of treatment of pulpitis or root canal infection. A reagent for detecting oral bacteria by the detection method according to claim 1 , comprising a reagent containing bismuth and homocysteine and a reagent containing bismuth and cysteine. The reagent according to claim 6 , further comprising a surfactant. A kit for diagnosing halitosis, periodontal disease, pulpitis or root canal infection, comprising a collecting means for collecting a sample from the oral cavity and the reagent according to claim 6 or 7 .

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