TWI424063B - Oral care methods and systems - Google Patents

Description

Oral health care method and system

The present application claims priority to U.S. Patent Application Serial No. 61/027,437, filed on Feb. 9, 2008. U.S. Patent Application Serial Nos. 61/027,432, 61/027, 431, 61/ 027, 420, and 61/ 027, 435, filed on Jan. 8, the entire contents of which are hereby incorporated by reference.

The present invention relates to a method for measuring the relative amount of caries and bacteria decomposing arginine in a mouth using a composition containing a free or salt type basic amino acid (for example, as part of a dental care method).

Arginine and other basic amino acids have been suggested for oral care and are believed to have significant benefits in combating tooth decay and tooth sensitivity. Commercially available arginine toothpaste containing CaviStat DenClude With ProClude (It contains arginine and calcium bicarbonate).

The type of biological flora in the mouth usually plays an important role in the formation of cavities and oral hygiene. For example, it is generally assumed that an important factor in the beneficial effects of arginine is that it and other basic amino acids can be metabolized by specific species of bacteria [eg, S. sanguis ], and that these bacteria do not produce dental caries and Caries-causing bacteria [eg S. mutans ] compete for position on the teeth and in the mouth. Bacteria decomposing arginine can use arginine and other basic amino acids to produce ammonia, thereby increasing the pH of the environment; while caries bacteria metabolizing sugar produces lactic acid, tending to lower the pH of plaque and remove teeth The minerals that eventually cause tooth decay.

It would be useful to have an effective method to monitor the type of biological flora in the mouth, for example, to determine the optimal treatment and to monitor the effectiveness of the patient's treatment.

The present invention provides a rapid and simple method for assessing bacterial flora in the mouth.

In a first embodiment, the invention measures plaque ammonia production to determine the relative population of bacteria decomposing arginine.

In another embodiment, the invention determines the amount of plaque lactic acid to determine the relative population of cariogenic bacteria.

In another embodiment, the invention uses polymerase chain reaction (PCR) (eg, quantitative real-time PCR) to identify a bacterial flora in the mouth (eg, in plaque or saliva).

In another example, the invention uses reverse transcriptase PCR (RT-PCR) to identify a bacterial flora in the mouth (eg, in plaque or saliva).

In another embodiment, an antibody probe (eg, a fluorescent antibody probe) is used to identify a bacterial flora in the mouth (eg, in plaque or saliva).

For example, the invention quantifies the amount of at least one cariogenic bacteria (eg, Streptococcus mutans) and at least one bacteria that break down arginine (eg, Streptococcus sanguis).

In another embodiment, the patient is assessed using one of the methods described above and the treatment is indicated accordingly.

The method of the invention is particularly useful for detecting potentially harmful changes in the plaque ecology, and for corrective treatment prior to measurable or significant demineralization or injury to the teeth.

The invention thus provides a method of improving oral health, for example

a. reducing or inhibiting the formation of dental caries;

b. reducing or inhibiting demineralization of teeth and promoting mineralization;

c. treating, reducing or inhibiting early enamel lesions;

d. reduce the allergic reaction of the teeth;

e. reduce or inhibit gingivitis;

f. promote the healing of ulcers or wounds in the mouth;

g. reduce the amount of acid-producing bacteria;

h. increase the relative amount of bacteria decomposing arginine;

i. inhibiting the formation of microbial biofilms in the oral cavity;

j. increasing and/or maintaining the plaque pH to at least pH 5.5 after stimulation with sugar;

k. reduce plaque accumulation;

l. Treat, alleviate or reduce dry mouth;

m. whitening teeth;

n. Promote general health, including cardiovascular health (eg, the use of reduced likelihood of systemic infection through oral tissues);

o. Immunize the teeth against caries bacteria and their effects;

p. cleaning teeth and mouth and / or

q. Reduce tooth corrosion;

The method comprises measuring the bacterial flora of the oral cavity (e.g., using any of the methods described above) and, if necessary, administering an oral health care product comprising an effective amount of a basic amino acid (e.g., arginine) or a salt thereof.

The invention further provides the use of a basic amino acid in free or salt form for the manufacture of a medicament according to the method of the invention, wherein the oral biobacteria contain an increase in the amount of cariogenic bacteria and/or an increase in the amount of lactic acid, and/or decomposition Patients with low levels of amino acid bacteria and/or low plaque ammonia production improve oral health.

The invention further provides a method of enhancing the aesthetics of the mouth (wherein enhancing aesthetics may include, for example, making the teeth whiter and/or reducing bad breath), the method comprising measuring the oral flora using the method according to the invention, if the caries bacteria are indicated Increased volume and / or increased lactic acid, and / or decreased bactericidal bacteria and / or decreased plaque ammonia production, oral health care containing free or salt basic amino acid product.

Plaque metabolism - ammonia production

The ability of plaque to convert arginine to ammonia is an indicator of the activity of arginine. Some bacteria have the ability to convert arginine to ammonia, just as some bacteria convert sugars into acids. It is advantageous to increase the relative concentrations of the lysine-degrading strains because they produce conditions that are detrimental to the proliferation of caries-producing bacteria, which favor acidic conditions and increase the risk of tooth decay. The use of arginine per day is expected to produce plaque ecological transfer that facilitates the breakdown of arginine bacteria in a manner similar to the condition in which regular intake of sugar produces conditions conducive to acidogenic bacteria. Ammonia is a neutralizing acid base that helps maintain the pH of neutral plaque. Neutral pH conditions are beneficial for non-pathogenic bacteria. The real-time PCR method for determining metabolically active (live) and inactivated (dead) bacteria is determined by measuring the concentration of the selected arginine-degrading bacteria (as described further below), and the amount of ammonia produced can be measured to determine the conversion of spermine. The contribution of all bacteria to which the acid is ammonia.

Ammonia test kits are commercially available, for example, from Diagnostic Chemicals Limited (Oxford, CT), which can measure the amount of ammonia produced. The principle of quantification and determination is known as ammonia and alpha-ketoglutaric acid and reduced smoke. Alkaline guanamine adenine dinucleotide phosphate (NADPH) reacts to form L-glutamic acid and NADP; this reaction is catalyzed by glutamate dehydrogenase (GLDH); the absorbance at 340 nm is reduced with ammonia due to NADPH oxidation The concentration is proportional. After the predetermined treatment procedure, plaque samples were collected. In several applications, plaque is obtained from enamel or HAP samples mounted on a retainer. In other applications, plaque is obtained directly from the teeth.

Plaque ecology based on the amount of lactic acid

Just as measuring the amount of ammonia as a proxy for decomposing the amount of arginine bacteria, lactic acid is a proxy for the amount of caries caused by caries. Patients who took plaque sampling did not eat or drink water the night before, and did not brush their teeth in the morning; rinsed with 10% sucrose solution for 2 minutes; after 8 minutes, the tooth surface was scraped off and plaque was collected. The plaque samples were collected in pre-weighed tubes placed on ice and the plaque weight was measured. The analysis included the addition of ice-cold water to a known plaque sample, followed by heating the sample to 80 ° C for 5 minutes to kill the bacteria and release all of the acid, and then the sample was placed in ice water for 5 minutes. The sample was then centrifuged and the supernatant was filtered. The concentration of lactic acid was determined by capillary electrophoresis.

Plaque ecology based on quantitative real-time PCR

Quantitative real-time PCR (PCR) is a highly sensitive method for quantifying DNA. Since the amount of DNA is directly related to the amount of bacteria present, the total bacterial amount is quantified using bacterial DNA isolated from dental plaque. Real-time PCR is recognized as a powerful and sensitive technology by government agencies such as the Center for Disease Control and the FDA. Probes are designed to detect the total amount of oral bacteria or specific bacteria such as Streptococcus mutans or Streptococcus sanguis using known gene sequences of many oral bacteria. DNA isolated from dental plaque or saliva samples is amplified by polymerase chain reaction. The amount of DNA increases exponentially with each PCR reaction cycle. This technique is referred to as "instant" because the reaction is performed immediately after the use of fluorescent reporter molecules. In one embodiment of the invention, SYBR Green is used as a reporter molecule. This molecule strongly fluoresces when it is combined with double-stranded DNA. The quantification is achieved by setting the fluorescence threshold and using the DNA standards of various concentrations to determine the number of cycles required to reach the threshold. The more DNA that is present, the less the number of DNA cycles required to reach a critical value. Commercially available real-time PCR instruments are commercially available from a number of manufacturers, such as Roche Diagnostics.

Plaque samples were obtained from enamel or hydroxyapatite samples of known and fixed surface area. The standardization of plaque collection is quite critical because the amount of DNA present is directly related to the amount of plaque collected. The method of normalizing the total bacterial amount using the plaque amount as a real-time PCR assay is not appropriate because the two quantities are significantly correlated. The results were recorded as micrograms of DNA per milliliter. Statistical analysis can be performed on DNA concentration or Ln (DNA concentration). The total bacterial count was analyzed by a two-factor variance analysis (ANOVA) using the patient and the treatment as a factor. If the difference detects a 95% confidence criterion, then the difference is considered significant. For specific bacteria such as Streptococcus mutans or Streptococcus sanguis, a two-factor variance analysis was performed using total bacterial amount as a cross-variable. The total amount of specific bacteria is related to the total bacterial population, so it is a more appropriate indicator of plaque ecological health.

In a specific embodiment of the invention, Streptococcus mutans is measured as an indicator of caries resistance; Streptococcus mutans is selected as a recognized risk factor associated with the initiation of tooth decay. Although other acid-producing bacteria involve the process of tooth decay, it is generally known that Streptococcus mutans plays an important role especially at the beginning and early stages of the cariogenic process. In a specific embodiment of the present invention, Streptococcus sanguis is selected as an indicator for transferring to a healthier plaque ecology; since Streptococcus mutans is generally known to have high decomposition arginine activity (the ability to convert arginine to ammonia) bacterial.

Plaque ecology according to RT-PCR

Reverse transcription PCR was used to determine RNA transcripts in samples. The RNA is isolated, the transcript is converted to cDNA using reverse transcriptase, and the cDNA is amplified using PCR. The advantage of RT-PCR is that the oral bacterial detection method of the DNA system cannot detect the viability of these strains. Because oral bacteria are most commonly found in biofilm communities, the DNA of dead bacteria can remain in the biofilm structure for long periods of time after death. Other methods, such as the Live Dead kit (Molecular Probes), can detect whether a organism has compromised membranes, but cannot directly detect a particular species.

Therefore, the reverse transcription real-time PCR method is a method for quantifying a living organism of a specific oral bacterial strain in a complex colony. mRNA has a fairly short half-life and is therefore an indicator of newly viable bacteria. Applicants and the like have developed a strain-specific primer for the elongation factor tuf (elongation factor tuf ). This gene is not significantly regulated by growth stages, media or environmental conditions, thus minimizing the virtual effects of the number of bacteria detected. Using Aggregatibacter actinomycetemcomitans as a test organism, survival differences can also be detected in a mixed population of live bacteria and bacteria killed by EtOH in the presence of as little as 20% of living organisms. In addition, this method reliably identifies the presence of Actinobacillus actinomycetes in a mixed population of up to six different bacterial species. The calculated bacterial concentration was closely related to the value estimated from the OD _{610 of the} same culture (r = 0.96, difference < 1%). This assay represents a method of studying the ecology of a particular organism in a complex oral environment. When a further genetic sequence data is available, a broad variety of oral bacteria can be developed.

Bacterial amount based on fluorescent antibody probe

The amount of caries-type bacteria (for example, Streptococcus mutans) and/or non-carious bacteria (for example, Streptococcus sanguis) in saliva is detected by using a monoclonal antibody using a dental caries diagnostic kit. The specific antibody used is specific to the bacterial species and has fluorescence attached to the antibody. The amount of bacteria can be detected by measuring the amount of fluorescence emitted.

Instance Example 1 Real-time PCR for measuring the total amount of plaque bacteria

The total amount of plaque bacteria in the patient (microgram bacterial DNA/ml) was measured using various toothpaste formulations, as described previously:

The arginine-fluoride formulation effectively reduces the total bacterial plaque load and the plaque load of Streptococcus mutans (cavity) and increases the load of Streptococcus sanguis (decomposed arginine).

Example 2 - Ammonia production

Ammonia production is measured using a variety of toothpaste formulations, as described above:

The amount of ammonia produced in plaque of patients using arginine-containing formula was significantly higher.

Example 3 - Amount of lactic acid

The plaque lactic acid amount of the patient was measured by capillary electrophoresis as described above, and the results showed that lactic acid was significantly increased in the presence of sucrose.

Example 4 - Real-time PCR / RT-PCR

The invention combines the principle of instant PCR detection of bacterial strains by using messenger RNA (mRNA) as an indicator of intracellular biological activity. After purification of mRNA from bacterial samples, reverse transcription real-time PCR is used to detect and quantify specific bacteria in a simple or complex environment. The present invention encompasses sequences of primers as well as mRNA identification methods and uses thereof.

One of the functions of DNA in living cells is to encode to synthesize proteins. The DNA encodes its corresponding mRNA strand, which is then used as a guide for assembling the final protein. Unlike DNA, mRNA has a very short half-life (seconds to minutes) and is present only in cells that are living or just dead; DNA is present in cells in a fixed set, and the amount of mRNA often changes in response to conditions in which cells survive. The performance of different proteins may be adjusted upwards or downwards in response to temperature changes, growth media, growth stages and other environmental conditions. Therefore, if the target gene is not carefully selected, changes in environmental conditions are likely to be misinterpreted as changes in the viability of the ethnic group. In order to avoid these effects, the present invention uses an elongation factor tu, its gene tuf , as a target sequence. This sequence has previously been used as a marker because tuf performance is little or no change under different experimental conditions.

The real-time PCR uses a basic chemistry that supports the amplification of genetic material by polymerase chain reaction (PCR) and is coupled to a real-time detection of fluorescent markers (which is a mechanism for quantifying the number of sets of established gene sequences present after each amplification cycle). The simplest of these methods is the use of SYBR Green I, a fluorescent probe specifically embedded in double-stranded DNA (dsDNA); thus the amount of SYBR Green fluorescence increase is associated with higher dsDNA concentrations. When this dye is incorporated into a PCR reaction that primarily uses a particular gene sequence, the increase in fluorescence is equivalent to an increase in the number of sets of target genes. Subsequently, the number of cycles in which the signal crosses the predetermined intensity threshold is related to the concentration of the gene sequence in the starting material.

The development of real-time PCR technology has enabled it to detect and quantify specific biological species quickly and with high precision. Conventional methods for quantifying bacterial species rely on the development of primers encoding the variable regions of the 16s ribosomal subunit DNA. This unit is critical for bacterial replication and, therefore, its sequence is not susceptible to mutation. Detection of specific 16s rDNA sequences for specific species contributes to the detection and enumeration of single bacterial species in complex environments.

The design of the primers is based on the sequences of the tuf genes obtained from the National Center for Biotechnology Information and the Los Alamos Oral Pathogens Database. The sequences were arranged using the DNA Star Lasergene program MegAlign standard elements. This arrangement is used to select regions with greater tillering and to maximize the similarity of species specificity. The selection of primer sequences was based on information analysis from the Roche Diagnostics LightCycler Probe Design software. The primers covered by the present invention include not only those who have been designed and tested, but also all primers for this gene site of oral pathogens.

Any RNA can be isolated from the sample using an appropriate RNA detachment kit or other RNA detachment method; any preferred method of singly RNA can be used. The purified RNA is treated twice with the appropriate DNase treatment reagent; this step degrades any contaminating DNA in the RNA preparation and prevents false positive results. The isolated RNA is then reverse transcribed to generate complementary DNA (cDNA) molecules; the resulting cDNA is amplified and detected using SYBR Green. An immediate PCR reaction without a reverse transcription step can be performed to control the quality of the DNA completely removed; the PCR product obtained by the lack of reverse transcription reaction must be the result of contaminating the DNA.

An immediate reverse transcription PCR reaction was performed on RNA samples isolated from cultures containing known amounts of live bacteria to generate a standard curve. The known concentration of the second derivative of each known sample is plotted against its known concentration of bacterial cells. The second derivative maximum of the RNA amplification curve isolated from the unknown bacterial sample can then be compared to the standard curve to determine the concentration of living organisms in the sample population. This data will be valuable information to curb the effects of antibacterial and active molecules on the environmental ecology of the oral cavity.

The following primer pairs were designed to amplify a 228 base pair region derived from the Tuf gene of Actinobacillus symbiosis:

These primers are used to amplify RNA from the pure culture of Actinobacillus compositae and the mixed populations containing or not containing Actinomyces elegans; the result, especially between fluorescence (F1) and circulation number The relationship is shown in Figure 1. In Fig. 1, "water" represents the negative control group of purely symbiotic A. faecalis culture and "Aa" is its positive control group; "Mix 1" is self-contained Prevoltella intermedia , distant chain The populations of Streptococcus sobrinus , Streptococcus oralis , and Actinomyces viscosus are purified, so amplification using their primers should be negative; "Mix 3" is derived from Actinobacillus elegans , Porphyromonas gingivalis , Streptococcus gordonii , Streptococcus mutans, and Streptococcus sanguis, the amplification of Actinobacillus actinomycetes should be positive.

This graph demonstrates that while the mixed population containing Actinobacillus actinomycetes is amplified in a similar curve to the positive control group, the mixed population lacking Actinomyces elegans follows the same amplification curve as the water control group; Primers can accurately detect Actinobacillus elegans from various RNA mixtures.

The ability of these primers to accurately detect and quantify only the live line A. faecalis organisms can be determined as described below. The known concentration of Actinobacillus actinomyces cells was suspended in 80% ethanol for 15 minutes for sterilization. The bacteria were then pelleted by centrifugation and the pellet was resuspended in fresh Brain Heart Infusion liquid growth medium. The ethanol-killed bacteria were incubated overnight at 37 ° C, and their growth was examined to confirm the retention of inactive organisms. The ethanol-killed bacteria are then mixed with the living organism at a predetermined ratio for reverse transcription and subsequent real-time PCR. Amplification of these samples is shown in Figure 2, which shows the amplification of RNA from a mixed population of live and dead line A. faecalis.

Although all populations used as templates for this reaction contained the same total amount of organisms, earlier amplification was observed in samples containing more living organisms, indicating that the assay can be used in a mixed population of live and dead bacteria. Living organisms are detected. In addition, the melting curves of their samples, as shown in Figure 3, show that a single, identical product was amplified in all samples, demonstrating the high specificity of the assay. Figure 3 shows the melting peak analysis of products amplified from pure line A. symbiosis and mixed cultures. The overlap of these curves indicates that a single product is amplified in all samples. Table 2 shows the comparison of predicted and calculated values of the biological numbers in the selected standard curve samples.

Calculate the approximate number of living organisms in each ethnic group based on the known concentration of live and killed starting cultures, and use in combination with the second derivative maximum of each amplification curve to generate a standard curve; the results are shown in Figure 4. Shown, it illustrates the standard curve generated from the amplification of known concentrations of live and dead line A. faecalis and the linear regression of the standard curve; the r ² value of the regression line is 0.96.

The r ² value of the linear regression line indicates the proximity of the regression equation to the observed values. An r ² close to 1.00 indicates that the observations closely match the regression line. With respect to the above example, the standard curve has an r ² value of 0.96, indicating that approximately 96% of the total variation observed in this line is due to the actual measured variation in the sample, so this standard curve can be used to calculate the concentration of living organisms in the unknown population.

In fact, in a single experiment, the concentration of living organisms calculated from this standard curve was not significantly different from the actual concentration added prior to RNA detachment, and the difference was <20%. According to their data, this assay represents a rapid and accurate method for detecting and quantifying living organisms in specific strains in complex biota; a potentially powerful tool for analyzing the effects of various treatments on oral microbial ecology.

Figure 1 Amplification of Actinobacillus actinomycetemii from pure cultures and mixed bacterial populations.

Figure 2 Amplification of RNA from a mixed population of live and dead line symbiosis A. faecalis.

Figure 3 is a melting peak analysis of products amplified from pure line A. faecalis and its mixed cultures.

Figure 4 is a standard curve generated from amplification of a known concentration of live and dead line symbiotic Actinobacillus and a linear regression of the standard curve.

Claims (7)

A method for assessing an oral biological flora, the method comprising measuring the amount of bacteria decomposing arginine and measuring the amount of cariogenic bacteria, wherein the amount of the degrading arginine bacteria is evaluated by measuring the amount of plaque ammonia produced, and The amount of the cariogenic bacteria is evaluated by measuring the amount of lactic acid produced, and wherein the amount of the lysine-degrading bacteria and the amount of the cariogenic bacteria are quantitative real-time PCR, quantitative RT-PCR, and/or fluorescent antibody detection. Needle to quantify. The method of claim 1, wherein the lysine-degrading bacterium comprises S. sanguis . The method of claim 1, wherein the cariogenic bacteria comprises S. mutans . The method of claim 1, wherein the method is for detecting potentially harmful changes in the plaque ecology prior to measurable or significant demineralization or injury to the teeth. Use of a basic amino acid or a salt thereof for the manufacture of an oral health care product for use in a dental organism comprising a cariogenic bacteria measured by the method according to any one of claims 1 to 4 An increase in the amount and/or an increase in the amount of lactic acid, and/or a decrease in the amount of arginine bacteria and/or a decrease in the amount of plaque ammonia production: a. reduction or inhibition of the formation of dental caries; b. reduction or inhibition of demineralization of the teeth To promote and promote mineralization; c. treat, reduce or inhibit early enamel lesions; d. reduce allergic reactions to the teeth; e. reduce or inhibit gingivitis; f. promote the healing of ulcers or wounds in the mouth; g. reduce the amount of acid-producing bacteria; h. increase the relative amount of bacteria that break down arginine; i. Forming a microbial biofilm; j. increasing and/or maintaining plaque pH to at least pH 5.5 after sugar stimulation; k. reducing plaque accumulation; l treating, alleviating or reducing dry mouth; m. whitening To promote general health, including cardiovascular health; o. protect teeth against or against immune to caries; p. clean teeth and mouth and / or q. reduce tooth corrosion. The use of the fifth aspect of the invention, wherein the basic amino acid is arginine or a salt thereof. Use of a basic amino acid or a salt thereof for the manufacture of an oral health care product comprising an increase in the amount of cariogenic bacteria in an oral biological flora measured by the method according to any one of claims 1 to 4 And/or an increase in the amount of lactic acid, and/or a decrease in the amount of bacteria degrading arginine and/or a decrease in the amount of plaque ammonia produced to improve oral aesthetics.