CA2711265A1 - Assessment of oral cavity bioflora - Google Patents
Assessment of oral cavity bioflora Download PDFInfo
- Publication number
- CA2711265A1 CA2711265A1 CA2711265A CA2711265A CA2711265A1 CA 2711265 A1 CA2711265 A1 CA 2711265A1 CA 2711265 A CA2711265 A CA 2711265A CA 2711265 A CA2711265 A CA 2711265A CA 2711265 A1 CA2711265 A1 CA 2711265A1
- Authority
- CA
- Canada
- Prior art keywords
- levels
- bacteria
- plaque
- reduce
- cariogenic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
- A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/18—Dental and oral disorders
Abstract
This, intention relates to methods of assessing the biofiora of the mouth and of providing appropriate treatment utilizing a basic amino acid in accordance with the assessment.
Description
ORAL CARE 'METHODS AND SYSTEMS
[00011 This application claims the benefit of United States Patent Application Serial No, 61/027A37 filed February 9, 2008, and also claims the benefit of United States Patent Application Serial No. 61/027,442 filed February 9, 2008, and United States Patent Application Serial Nos. 6l/027,432; 61/02:,431; 611027,420; and 61/027,435 all filed February 8. 2008, the contents of which applications are all incorporated herein by reference.
FIELD OF THE INVENTION
100031 This invention relates to methods of measuring relative levels of cariogenic and arginolytic bacteria in the mouth, e.g., as part of a dental care regimen using compositions comprising a basic amino acid in free or salt form.
BACKGROUND OF THE INVENTION
100041 Arginine and other basic amino acids have been proposed for use in oral care and are believed to have significant benefits in combating cavity formation and tooth sensitivity.
Commercially available argini.ne-based toothpastes are DenClude and ProClude containing CaviStat , which contain arginine and calcium bicarbonate.
100051 The type of bioflora in the mouth plays a significant role in the development of cavities and in oral health generally. For example, it has been hypothesized that a significant factor in the beneficial effect of arginine is that arginine and other basic amino acids can be metabolized by certain types of bacteria, e.g., S. sanguis which are not cariogenic and which compete with cariogenic bacteria such as S. inutuns, for position on the teeth and in the oral cavity. The arginolytie bacteria can use arginine and other basic arnino acids to produce ammonia, thereby raising the pH of their environment, while cariogenic bacteria metabolize sugar to produce lactic acid, which tends to lower the plaque pH and demineralize the teeth, ultimately leading to cavities [00061 It would be useful to have an efficient way to monitor the type of bioflora in the mouth, e.g., to determine the optimal treatment and to monitor the effectiveness of treatment of patients.
Bail 1 T..._ I i 7H,)Ci~.i ven1l n pr': and the biotio%L...
mouth.
100081 In a first embodiment, the invention a ae r . plaque ammonia p.ud .
'icon levels to determine the relative population of arginc lytic ba~tcria.
[00091 In another embodiment, the invention measures plaque lactic acid levels to determine the relative population of cariogenic bacteria.
100101 In another embodiment, the invention uses the polymerise chain reaction (PCR), for example quantitative real time PCR, to characterize the bioflora in the m Louth, e.g., in the plaque or saliva.
100111 In another example, the invention uses reverse transcriptase PCR (RT-PCR) to characterize the bioflora in the mouth. e.g., in the plaque or saliva.
100121 In another embodiment, antibody probes. e.g., fluorescent antibody probes are used to characterize the bioflora in the mouth, e.g., in the plaque or saliva.
100131 For example, the invention quantifies levels of at least one cariogenic bacteria, e.g., S. ratans. and at least one arginolytic bacteria, e.g., S. san uis.
[00141 In another embodiment, the patient is assessed using one of the foregoing methods, and treatment prescribed accordingly.
[[0151 The methods of the invention are particularly useful to detect potentially damaging changes in plaque ecology and to allow corrective treatment before there is measurable or significant demineralization or damage to the teeth.
100161 The invention thus provides methods to enhance oral health., e.g., to a, reduce or inhibit formation of dental caries, b. reduce or inhibit demineralization and promote remineralization of the teeth, c. treat, reduce or inhibit formation of early enamel lesions, d. reduce hypersensitivity of the teeth, e. reduce or inhibit gingivitis, f promote healing of sores or cuts in the mouth, g. reduc_c 1 , c1s of acid producing bact ri: , h. ti t i u at .,~ .. ~ i .
challenge.
k. reduce plaque accumulation, 1. treat, relieve or reduce dry mouth, m. whiten teeth, n. enhance systemic health, including cardiovascular health, e.g., by reducing potential for systemic infection via the oral tissues, o. immunize the teeth against cariogenic bacteria and their effects, p. clean the teeth and oral cavity and/or q. reduce erosion of the teeth comprising measuring the bioflora of the oral cavity. e.g., using any of the foregoing methods, and if indicated, administering an oral care product comprising an effective amount of a basic amino acid or salt thereof, e.g., arginine.
100171 The invention further provides the use of a basic amino acid, in free or salt form, for the manufacture of medicament for enhancing oral health in a subject whose oral cavity bioflora comprise elevated levels of cariogenic bacteria and/or elevated lactate levels, and/or low levels of arginolytic bacteria and/or low levels of plaque ammonia production, as measured by a method according to the present invention.
[00181 The invention further provides a method for cosmetically enhancing the oral cavity (wherein such cosmetic enhancement may include e.g. making teeth whiter and/or reducing halitosis) which method comprises measuring the bioflora of the oral cavity using a method according to the present invention, and if indicated by the presence of elevated levels of cariogenic bacteria and?`or elevated lactate levels, and/or the presence of low levels of arginolytic bacteria and/or low levels of plaque ammonia production, administering an oral care product comprising a basic amino acid in free or salt form.
DETAILED DESCRIPTION
Plaque Metabolism Ammonia Production [00191 The ability of dental. plaque to conc.: -trine to E y. - -Ii _.-iv ty.
Certain bacteria have the ability to convert ar ins: to anti a. just as n l . _::a _~.r ~ tt acad. it is ` ef:cia.l tc i _ r e rc? Ld . _.
nc ntrati ..: t l li.' 1' ' .,tit ;l .creri t crcute L: 't!
unfavorable. for prolifcrat',,n t e/ c i'.C l c which favor ac .ic 4o -,luil:o , and increase caries risk. Daily use of arginine is expected to create a shift in the plaque ecology.
that favors arginolytic bacteria in an analogous manner that frequent consumption of sugar creates conditions that favor acid producing bacteria. Ammonia is a base that is capable of neutralizing acids and helps maintain neutral plaque pH. Neutral pfl conditions are more i:t\ orable to nonpathogenic bacteria. Measurement of ammonia production measures the contribution from all the bacteria capable of converting arginine to ammonia.
This is in contrast to the real time PCR method (further described below) which measures concentration of select arginolytic bacteria and does not distinguish between metabolically active (live) and inactive (dead) bacteria.
100201 Ammonia detection kits are available commercially, e.g., from Diagnostic Chemicals Limited (Oxford, CT) to measure ammonia production. The principle for the quantification and determination is that ammonia is known to react with alpha-ketoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form L-glutamate and NADP. The reaction is catalyzed by glutamate dehydrogenase (GLDH). The decrease in absorbance at 340 nn due to the oxidation of NADPH is proportional to the ammonia concentration. Plaque samples are collected after a predefined treatment protocol. In some applications, plaque is harvested from enamel or HAP specimens mounted on a retainer. In other applications, plaque is harvested directly from the teeth.
Plaque ecolosp by Lactic Acid Levels 100211 Just as the measurement of ammonia levels serves as a proxy to measure the levels of arginolytic bacteria, lactic acid serves as a proxy to measure the levels of cariogenic bacteria. Subjects have plaque taken without morning oral hygiene and without eating or drinking from the previous evening. They rinse with a 10% sucrose solution for 2 minutes.
After 8 minutes, plaque is collected by scraping the tooth surface(s). Plaque samples are collected on ice in preweighed tubes, and the plaque weight is determined. The analysis includes adding ice cold water to the known amount of plaque samples then heating the samples to 80 dey C for 5 minutes to kill the bacteria and to release all acids before the I. :l .' minutes. T 'he samples are h r c'entrifbged water for a measured us' ~ .' -' /C que - !>' t aniiiaiivÃ' Refit lime P( 100221 Qt,i i .five real time PCR (Polymerase Chain. Reaction) is a highly means of quantifying DNA, Bacterial DNA isolated from dental plaque is used to quantify the total levels of bacteria since the amount of DNA is directly related to the amount bacteria present. Real time PCR is recognized by government organizations such as the Center for Disease Control and the :FDA as a very powerful and sensitive technique.
Taking advantage of the known genomic sequence of many oral bacteria, probes are designed to detect total levels of oral bacteria or specific bacteria such as S. mutans or S. sanguis.
DNA isolated from the samples of plaque or saliva is amplified by the polymerase chain reaction.
The amount of DNA increases exponentially with each cycle of the PCR reaction. The technique is referred to as "real time" because the reaction is followed in. real time through the use of fluorescent report molecules. In one embodiment of the invention, SYBR Green is used as the reporting molecule. This molecule fluoresces strongly upon coordination with double stranded DNA.
Quantification is achieved by setting a fluorescent threshold and using DNA
standards at various concentrations to determine the number of cycles needed to reach the threshold. The more DNA present, the smaller number of DNA cycles are needed to reach the threshold.
Commercial Real Time PCR instruments are available from numerous manufacturers, such as Roche Diagnostics.
[0023] Plaque samples are harvested from enamel or hydroxyapatite specimens with known and constant surface area. Standardization of plaque collection is critical because the amount of DNA present is directly related to how much plaque is collected. It is inappropriate to use plaque mass as a means standardizing total bacteria measured by real time PCR
because the two quantities are significantly correlated. The results reported as ug DNA per ml. Statistics can be performed on the DNA concentration or Ln(DNA
concentration). For total bacteria, a two factor ANOVA is performed using the subject and treatment as factors.
Differences are considered significant if a difference is detected a 95%
confidence level. For specific bacteria such as ,S'. mutans or S. sunquis, a two factor ANCOVA is conducted using the total bacteria as the covariate. The total amount of specific bacteria as it relates to the total bacterial population is a more relevant marker of plaque ecology health.
1.00241 In a particular emnbc.hnic in unt: in, S. ors is measurer ik,er for ~. c.. factor YTdtiti73's`~ ~ t;i t,' _, L ~1! 1_.,- risk cto-the 1 are involved ai ÃSw i:arie=' } ' t = ->~. 1:wn to pl:.'; , I ri rl in the i i.`iation aT"id stages C,.- c J 1 ( k. 1 1 ,; process. In one i i : C.iventlon. . is cho :i as a marker fir r a shift to healthier plaque ec = . because S. sanguis is known to exhibit a high level of arginolytic activity (ability to convert arginine to ammonia).
Plaque ecology by RT-PC'R
100251 Reverse transcription PCR measures RNA transcripts in a sample. The RNA
is isolated, the transcripts converted to cI)NA using reverse transcriptase. and the cI)NA is amplified using PCR. The advantage of RT-PCR is that DNA-based methods for the detection of oral bacteria are unable to determine the viability of those species. Because oral bacteria are most often found in biofilm communities, the DNA of dead bacteria can be retained within the biofilm architecture for long periods of time following killing. Other methods, such as fluorescence-based viability assays (Live Dead kit, Molecular Probes), can detect whether or not organisms have compromised membranes, but do not directly detect specific species.
100261 Reverse transcription real time PCR is thus a method to quantify the viable organisms of a specific species of oral bacteria present within in a complex community.
mRNA has a relatively short half life and therefore is indicative of recently active bacteria.
We have developed species-specific primers to the elongation factor tiff This gene is not significantly regulated by growth phase, media or environmental conditions, thereby minimizing spurious effects on detected numbers of bacteria. Using Aggregatibacter actinomviceterncoritan.c as our test organism, viability differences in mixed populations of live and EtOH killed bacteria may be detected when as few as 20% of the organisms present are viable. Additionally, the method allows reliable identification of the presence of A.
a(.ctinnarntvcctcerncoinitarns in mixed species populations containing up to six different species of bacteria. Calculated bacterial concentrations correlated closely to values estimated based on ODh, Eor the same cultures (r= 0.96, <1% difference). This assay represents a means of studying the ecology of specific organisms within the complex environment of the oral cavity. As further genetic sequence data becomes available, primers can be developed to a wide variety of oral bacteria.
B , terial Levels by Flaat)rc~:scent Antibody, Probe 1002'! A carie~ I [ is kit,, is a-.c'd to detect the level of << t= c)1 _ype Of 01-,"
c, _ ., S. i utans and a a non-c, t. -c _ i_,; type, e.g.. S. sanguis, iii a through the uz,s of i. t l an fxe parti,au' at Hodies used are specific for the species of bacteria aid ra \ e a fluor i'rt_ attache,) :ie antibody. The levels of bacteria can be detected by measuring the < -new I a i Fluor Cray fiat is emitted.
EXAMPLES
Example 1 Real time PCR to measure total plaque bacteria levels 100281 Levels of total plaque bacteria (micrograms bacterial DNA r1) in subjects is measured using different toothpaste formulations, using the procedures described supra:
Total bacterial DNA S. mutans DNA S. sanguis DNA
250 ppm fluoride 6.091 0.09622 1.126 formulation (control) 1450 ppm fluoride 6.018 0.09903 1.1.0 7 formulation Formulation having 3.781 0.05998 1.291 21V O arginine bicarbonate and 1450 ppm fluoride 100291 The arginine-fluoride formulation is effective to reduce total bacterial plaque loads, and S. rnutans (cariogenic) plaque loads, while enhancing S. sanguis (arginolytic) loads.
Example 2 -- Ammonia production (00301 Ammonia production is measured in subjects using different toothpaste forrtulatios, using the method described above:
Ammonia level (ppm) 250 ppm fluoride 1.97 formulation (control) 1450 ppm fluoride 1.79 I- _-,r; is i n having 2.77 r,e w:id 14450 1 or.1e 100311 A monia production is significantly higher in plaque of subjects using the arginine-containing formulation.
Example 3 Lactic acid levels 100321 Plaque lactic acid levels are measured in subjects using capillary electrophoresis as described above, showing that lactate is significantly increased in the presence of sucrose.
Sucrose Plaque Challenged Plaque Lactate 1.87 7.82 0.37 nmollmg) Example 4 - Real time PCR ! RT-PCR
100331 This invention combines the principles of real-time PCR detection of bacterial species with the use of messenger RNA (mRNA) as an indicator of biological activity within cells. Following purification of mRNA from a bacterial sample, reverse transcription real time PCR is used to detect and quantify specific bacteria within a simple or complex environment. The invention covers the sequence of the primers as well as the mRNA
identification method and its application.
100341 One function of DNA within viable cells is to code for the synthesis of proteins.
DNA codes for its corresponding mRNA strand which is then used as the instructions for assembling finished proteins. Unlike DNA, mRNA has a very short half life (seconds to minutes) and is only present in cells that are either viable or very recently killed. Whereas DNA is present in cells in a fixed number of copies, mRNA levels are often changed. in response to the conditions in which a cell exists. Expression. of different proteins may be up-or downregulated in response to temperature changes, growth media, growth phases and other environmental conditions. Therefore, ifthe target gene is not carefully chosen, it is possible that fluctuations in env-ironmrental condiE=on, ~%.:11 be falsely rà as population a the ' L cc i a~ y u d : t. c ' because ;ei # à r .et sequence- Th, 3 sequence ha as a Tl . n ',I tuf expression has been observed under different i n ntal conditions.
[00351 Real time PCR uses the basic chemistry behind polynaeras~ reaction (.PC'R) amplification of genetic material and couples it with real time detection k ,C
i1 zorescent labels as a mechanism of quantifying the number of copies of a given genetic sequence pr :c nt after each amplification cycle. The simplest of these methods uses SYBR Green I, a fluorescent probe that intercalates specifically into double stranded DNA (dsDNA).
Increasing levels of SYBR Green fluorescence therefore correlate to greater concentrations of dsDN
A. When this dye is included in a PCR reaction primed using specific genetic sequences, the increase in fluorescence corresponds to an increase in the number of copies of the target gene.
Subsequently, the cycle number at which the signal crosses a predetermined intensity threshold can be correlated to the concentration of the genetic sequence in the starting material.
100361 The development of real time PCR technology has made it possible to detect and quantify specific biological species rapidly and with a high degree of accuracy. Conventional methods for quantification of bacterial species rely on the development of primers to the variable region of the DNA encoding the 16s ribosomal subunit. This subunit is critical to bacterial replication and its sequence is, therefore, not readily mutated. The detection of 6s rDNA sequences specific to a particular species can facilitate the detection and enumeration of a single bacterial species within a complex environment.
100371 Primers are designed based on the sequences of wuf genes from publically available databases (National Center for Biotechnology Information. and the Los Alamos Oral Pathogens Database). Sequences are aligned using the DNA Star Lasergene program MegAlign module. This alignment is used to select a region of greater divergence in order to maximize the likelihood of species specificity. Primer sequences are selected based upon analysis information available from the Roche Diagnostics LightCycler Probe Design software. Primers covered by this invention include not only those already designed and tested, but all primers to this genetic region in oral pathogens.
100381 Total RNA is isolated from samples using an appropriate RNA isolation kit or other RNA isolation method. Any preferred method for RNA isolation can be used. Purified RNA is treated 2 times with appropriate DNase treatment reagents. This step degrades any DN' :R\; :)rep and `l l i I,I i i, },<i: is )sitives.
t. } 4 11s. T'he re .r` ) ieH: and 1 .
c,.-mr ol for the conl ,,-'C.Le Civic a real P01' reverse transcription step. PCK Jr,'i uct, obtained. in the abcLi, c of .1 reverse Iran reaction must be the result of acing DNA_ I
100391 A standard curve is generated by performing the real time reverse transcription PCR reaction on RN.A samples isolated from cultures containing known amounts of viable bacteria. The second derivative maximum value for each known sample is plotted against its known concentration of bacterial cells. The second derivative maximum of amplification curves of RNA isolated from unknown bacterial samples can then be compared to the standard curve to determine the concentration of viable organisms within the sample population. This data would be valuable information in deterring the effects of antibacterials and active molecules on the ecology of the oral environment.
100401 The following primer pair is designed to amplify a 228 base pair region of the trtf-gene from AggregatibactÃ:r (9.ctinohacilia s) actinomyceetemcomitans:
Primer Sequence T. ( C) %GC AG Annealing name (kcal.nlol) Temp.
Forward 5' AAGCGCGTGG rATCAC 3' 49.05 56.25 -27.56 55 C
Reverse 5' -- TG'TAAGGAACACCTA .-.._..-. 3' 31.52 40.00 -20.15 55 C
Table 1. Properties of primers designed for quantification of mRNA expression of tufin A.
actinomvicet (: Inc om itans .
100411 These primers are used to amplify RNA isolated from both pure cultures of A.
actinrnoyceterncomitans and mixed populations both with and without A.
aetinomviceterncomitans included. The results, in particular the relationship between fluorescence (Fl) and cycle number are shown in the graph of Figure 1. In Figure 1, "water"
represents the negative control and "Aa" is the positive control of pure A.
actinontycetetncomitans culture. "Mix 1" was purified from a population containing Prevo/tella interniedia, Streptococcus sobrinus, Streptococcus oralis, and Actinornyces tiiscosus and should, therefore be negative for amplification with these primers. "Mix 3" is from a population containing A. acctino tyc.,etemcÃ3nnitans, Por 3hyrom 3nas gingiralis, trervo oc cus cr, ~? C ??r7Zl. Streptococcus t? itans, and t rrptoc'occus sanquinrs and should be M0421 This I'-is ampl. f c i "'i; i .-:: 1.:r' curve o liicJ positive s r true iiu rn !..
,cJcing ii :.3lit}'Jv~ g ;t.' ::ITIi.:`.
amp are able to aL.
detect r 4.:'trl;: 3a J tan from within m 'x of RNV%
100431 The ability of these primers to accurately detect and quantify only viable .ac t$rarrrr~r c~c~t rr~t'omitrrrts organisms is determined as follows. A
known concentration of A.
aÃctinrormzycetWrrracornitcars cells are killed by suspending in 80% ethanol for 15 minutes. The bacteria are then pelleted by centrifugation and resuspended in fresh Brain Heart Infusion broth growth media. The ethanol killed bacteria are incubated overnight at 37 C and examined for growth to confirm that no viable organisms were remained. The ethanol killed bacteria are then mixed, in defined ratios, with viable organisms and reverse transcription followed by real time PCR is performed. The amplification of these samples is shown in Figure 2, which shows the amplification of RNA from mixes of live and dead A_ aetinomyeetenicornltan.s.
100441 Despite the fact that all of the populations used as templates for this reaction contained the same total number of organisms, earlier amplification is observed in samples containing more viable organisms, indicating that this assay is able to detect viable organisms within a mix of both live and dead bacteria. Additionally, the melting curve of these samples, as shown in Figure 3, indicates that a single, identical product is amplified in all samples, which demonstrates the high specificity of the assay. Figure 3 shows a melting peak analysis of products amplified from pure and mixed cultures of .1.
aetinomycetemcornitans. The overlap of these curves indicates that a single product is being amplified from all samples.
Table 2 shows a comparison of expected and calculated number of organisms in selected standard curve samples.
Sample Expected viable CFU Calculated viable CFU
100% 5.00 x 10 5.34 x 10' 50% 2.50 x 10' 2.28 x 10' 40% 2.00 x 107 2.54 x 10 30% 1.50x 10' 1.18x107 1.00 x 10 1.07 x 10' 5.00 x 106' 4 06 x 1 2.50x 10 .ic x T able 2. t c_, ud and caicLuated number ..x, _. ;: 'n selected standard Lars i 100451 Lased on the known concentrations of t le , . ,~,nd killed starting cultures, the approximate number of viable organisms in each popuiation is calculated and used in.
Ix conjunction with the second derivative maximum of each amplification curve to generate a standard curve. The results are shown in Figure 4, which illustrates a standard curve and linear regression of the standard curve generated from the amplification of known concentrations of viable and dead A. actimm L L ('-~~ .L.==rnitans. The r2 value of the regression line is 0.96.
100461 The r' value of a linear regression line indicates the closeness of fit of the regression equation to the observed values. An r2 closer to 1.00 indicates that the observed values match closely to the regression line. For the example above, the r-value of the standard curve is 0.96, indicating that about 96% of the total variation observed in the line is due to actual measured variation in the samples and that this standard curve can be used to calculate the concentration of viable organisms in unknown populations.
O0471 In practice, in a single experiment, the concentration of viable organisms calculated based on this standard curve is not significantly different from the actual concentration added prior to RNA isolation and differed by <20%. These data indicate that this assay represents a rapid, accurate means of detecting and quantifying viable organisms of a specific species within a complex population of organisms. This represents a potentially powerful tool for analyzing the effects of treatments on oral microbial ecology.
[00011 This application claims the benefit of United States Patent Application Serial No, 61/027A37 filed February 9, 2008, and also claims the benefit of United States Patent Application Serial No. 61/027,442 filed February 9, 2008, and United States Patent Application Serial Nos. 6l/027,432; 61/02:,431; 611027,420; and 61/027,435 all filed February 8. 2008, the contents of which applications are all incorporated herein by reference.
FIELD OF THE INVENTION
100031 This invention relates to methods of measuring relative levels of cariogenic and arginolytic bacteria in the mouth, e.g., as part of a dental care regimen using compositions comprising a basic amino acid in free or salt form.
BACKGROUND OF THE INVENTION
100041 Arginine and other basic amino acids have been proposed for use in oral care and are believed to have significant benefits in combating cavity formation and tooth sensitivity.
Commercially available argini.ne-based toothpastes are DenClude and ProClude containing CaviStat , which contain arginine and calcium bicarbonate.
100051 The type of bioflora in the mouth plays a significant role in the development of cavities and in oral health generally. For example, it has been hypothesized that a significant factor in the beneficial effect of arginine is that arginine and other basic amino acids can be metabolized by certain types of bacteria, e.g., S. sanguis which are not cariogenic and which compete with cariogenic bacteria such as S. inutuns, for position on the teeth and in the oral cavity. The arginolytie bacteria can use arginine and other basic arnino acids to produce ammonia, thereby raising the pH of their environment, while cariogenic bacteria metabolize sugar to produce lactic acid, which tends to lower the plaque pH and demineralize the teeth, ultimately leading to cavities [00061 It would be useful to have an efficient way to monitor the type of bioflora in the mouth, e.g., to determine the optimal treatment and to monitor the effectiveness of treatment of patients.
Bail 1 T..._ I i 7H,)Ci~.i ven1l n pr': and the biotio%L...
mouth.
100081 In a first embodiment, the invention a ae r . plaque ammonia p.ud .
'icon levels to determine the relative population of arginc lytic ba~tcria.
[00091 In another embodiment, the invention measures plaque lactic acid levels to determine the relative population of cariogenic bacteria.
100101 In another embodiment, the invention uses the polymerise chain reaction (PCR), for example quantitative real time PCR, to characterize the bioflora in the m Louth, e.g., in the plaque or saliva.
100111 In another example, the invention uses reverse transcriptase PCR (RT-PCR) to characterize the bioflora in the mouth. e.g., in the plaque or saliva.
100121 In another embodiment, antibody probes. e.g., fluorescent antibody probes are used to characterize the bioflora in the mouth, e.g., in the plaque or saliva.
100131 For example, the invention quantifies levels of at least one cariogenic bacteria, e.g., S. ratans. and at least one arginolytic bacteria, e.g., S. san uis.
[00141 In another embodiment, the patient is assessed using one of the foregoing methods, and treatment prescribed accordingly.
[[0151 The methods of the invention are particularly useful to detect potentially damaging changes in plaque ecology and to allow corrective treatment before there is measurable or significant demineralization or damage to the teeth.
100161 The invention thus provides methods to enhance oral health., e.g., to a, reduce or inhibit formation of dental caries, b. reduce or inhibit demineralization and promote remineralization of the teeth, c. treat, reduce or inhibit formation of early enamel lesions, d. reduce hypersensitivity of the teeth, e. reduce or inhibit gingivitis, f promote healing of sores or cuts in the mouth, g. reduc_c 1 , c1s of acid producing bact ri: , h. ti t i u at .,~ .. ~ i .
challenge.
k. reduce plaque accumulation, 1. treat, relieve or reduce dry mouth, m. whiten teeth, n. enhance systemic health, including cardiovascular health, e.g., by reducing potential for systemic infection via the oral tissues, o. immunize the teeth against cariogenic bacteria and their effects, p. clean the teeth and oral cavity and/or q. reduce erosion of the teeth comprising measuring the bioflora of the oral cavity. e.g., using any of the foregoing methods, and if indicated, administering an oral care product comprising an effective amount of a basic amino acid or salt thereof, e.g., arginine.
100171 The invention further provides the use of a basic amino acid, in free or salt form, for the manufacture of medicament for enhancing oral health in a subject whose oral cavity bioflora comprise elevated levels of cariogenic bacteria and/or elevated lactate levels, and/or low levels of arginolytic bacteria and/or low levels of plaque ammonia production, as measured by a method according to the present invention.
[00181 The invention further provides a method for cosmetically enhancing the oral cavity (wherein such cosmetic enhancement may include e.g. making teeth whiter and/or reducing halitosis) which method comprises measuring the bioflora of the oral cavity using a method according to the present invention, and if indicated by the presence of elevated levels of cariogenic bacteria and?`or elevated lactate levels, and/or the presence of low levels of arginolytic bacteria and/or low levels of plaque ammonia production, administering an oral care product comprising a basic amino acid in free or salt form.
DETAILED DESCRIPTION
Plaque Metabolism Ammonia Production [00191 The ability of dental. plaque to conc.: -trine to E y. - -Ii _.-iv ty.
Certain bacteria have the ability to convert ar ins: to anti a. just as n l . _::a _~.r ~ tt acad. it is ` ef:cia.l tc i _ r e rc? Ld . _.
nc ntrati ..: t l li.' 1' ' .,tit ;l .creri t crcute L: 't!
unfavorable. for prolifcrat',,n t e/ c i'.C l c which favor ac .ic 4o -,luil:o , and increase caries risk. Daily use of arginine is expected to create a shift in the plaque ecology.
that favors arginolytic bacteria in an analogous manner that frequent consumption of sugar creates conditions that favor acid producing bacteria. Ammonia is a base that is capable of neutralizing acids and helps maintain neutral plaque pH. Neutral pfl conditions are more i:t\ orable to nonpathogenic bacteria. Measurement of ammonia production measures the contribution from all the bacteria capable of converting arginine to ammonia.
This is in contrast to the real time PCR method (further described below) which measures concentration of select arginolytic bacteria and does not distinguish between metabolically active (live) and inactive (dead) bacteria.
100201 Ammonia detection kits are available commercially, e.g., from Diagnostic Chemicals Limited (Oxford, CT) to measure ammonia production. The principle for the quantification and determination is that ammonia is known to react with alpha-ketoglutarate and reduced nicotinamide adenine dinucleotide phosphate (NADPH) to form L-glutamate and NADP. The reaction is catalyzed by glutamate dehydrogenase (GLDH). The decrease in absorbance at 340 nn due to the oxidation of NADPH is proportional to the ammonia concentration. Plaque samples are collected after a predefined treatment protocol. In some applications, plaque is harvested from enamel or HAP specimens mounted on a retainer. In other applications, plaque is harvested directly from the teeth.
Plaque ecolosp by Lactic Acid Levels 100211 Just as the measurement of ammonia levels serves as a proxy to measure the levels of arginolytic bacteria, lactic acid serves as a proxy to measure the levels of cariogenic bacteria. Subjects have plaque taken without morning oral hygiene and without eating or drinking from the previous evening. They rinse with a 10% sucrose solution for 2 minutes.
After 8 minutes, plaque is collected by scraping the tooth surface(s). Plaque samples are collected on ice in preweighed tubes, and the plaque weight is determined. The analysis includes adding ice cold water to the known amount of plaque samples then heating the samples to 80 dey C for 5 minutes to kill the bacteria and to release all acids before the I. :l .' minutes. T 'he samples are h r c'entrifbged water for a measured us' ~ .' -' /C que - !>' t aniiiaiivÃ' Refit lime P( 100221 Qt,i i .five real time PCR (Polymerase Chain. Reaction) is a highly means of quantifying DNA, Bacterial DNA isolated from dental plaque is used to quantify the total levels of bacteria since the amount of DNA is directly related to the amount bacteria present. Real time PCR is recognized by government organizations such as the Center for Disease Control and the :FDA as a very powerful and sensitive technique.
Taking advantage of the known genomic sequence of many oral bacteria, probes are designed to detect total levels of oral bacteria or specific bacteria such as S. mutans or S. sanguis.
DNA isolated from the samples of plaque or saliva is amplified by the polymerase chain reaction.
The amount of DNA increases exponentially with each cycle of the PCR reaction. The technique is referred to as "real time" because the reaction is followed in. real time through the use of fluorescent report molecules. In one embodiment of the invention, SYBR Green is used as the reporting molecule. This molecule fluoresces strongly upon coordination with double stranded DNA.
Quantification is achieved by setting a fluorescent threshold and using DNA
standards at various concentrations to determine the number of cycles needed to reach the threshold. The more DNA present, the smaller number of DNA cycles are needed to reach the threshold.
Commercial Real Time PCR instruments are available from numerous manufacturers, such as Roche Diagnostics.
[0023] Plaque samples are harvested from enamel or hydroxyapatite specimens with known and constant surface area. Standardization of plaque collection is critical because the amount of DNA present is directly related to how much plaque is collected. It is inappropriate to use plaque mass as a means standardizing total bacteria measured by real time PCR
because the two quantities are significantly correlated. The results reported as ug DNA per ml. Statistics can be performed on the DNA concentration or Ln(DNA
concentration). For total bacteria, a two factor ANOVA is performed using the subject and treatment as factors.
Differences are considered significant if a difference is detected a 95%
confidence level. For specific bacteria such as ,S'. mutans or S. sunquis, a two factor ANCOVA is conducted using the total bacteria as the covariate. The total amount of specific bacteria as it relates to the total bacterial population is a more relevant marker of plaque ecology health.
1.00241 In a particular emnbc.hnic in unt: in, S. ors is measurer ik,er for ~. c.. factor YTdtiti73's`~ ~ t;i t,' _, L ~1! 1_.,- risk cto-the 1 are involved ai ÃSw i:arie=' } ' t = ->~. 1:wn to pl:.'; , I ri rl in the i i.`iation aT"id stages C,.- c J 1 ( k. 1 1 ,; process. In one i i : C.iventlon. . is cho :i as a marker fir r a shift to healthier plaque ec = . because S. sanguis is known to exhibit a high level of arginolytic activity (ability to convert arginine to ammonia).
Plaque ecology by RT-PC'R
100251 Reverse transcription PCR measures RNA transcripts in a sample. The RNA
is isolated, the transcripts converted to cI)NA using reverse transcriptase. and the cI)NA is amplified using PCR. The advantage of RT-PCR is that DNA-based methods for the detection of oral bacteria are unable to determine the viability of those species. Because oral bacteria are most often found in biofilm communities, the DNA of dead bacteria can be retained within the biofilm architecture for long periods of time following killing. Other methods, such as fluorescence-based viability assays (Live Dead kit, Molecular Probes), can detect whether or not organisms have compromised membranes, but do not directly detect specific species.
100261 Reverse transcription real time PCR is thus a method to quantify the viable organisms of a specific species of oral bacteria present within in a complex community.
mRNA has a relatively short half life and therefore is indicative of recently active bacteria.
We have developed species-specific primers to the elongation factor tiff This gene is not significantly regulated by growth phase, media or environmental conditions, thereby minimizing spurious effects on detected numbers of bacteria. Using Aggregatibacter actinomviceterncoritan.c as our test organism, viability differences in mixed populations of live and EtOH killed bacteria may be detected when as few as 20% of the organisms present are viable. Additionally, the method allows reliable identification of the presence of A.
a(.ctinnarntvcctcerncoinitarns in mixed species populations containing up to six different species of bacteria. Calculated bacterial concentrations correlated closely to values estimated based on ODh, Eor the same cultures (r= 0.96, <1% difference). This assay represents a means of studying the ecology of specific organisms within the complex environment of the oral cavity. As further genetic sequence data becomes available, primers can be developed to a wide variety of oral bacteria.
B , terial Levels by Flaat)rc~:scent Antibody, Probe 1002'! A carie~ I [ is kit,, is a-.c'd to detect the level of << t= c)1 _ype Of 01-,"
c, _ ., S. i utans and a a non-c, t. -c _ i_,; type, e.g.. S. sanguis, iii a through the uz,s of i. t l an fxe parti,au' at Hodies used are specific for the species of bacteria aid ra \ e a fluor i'rt_ attache,) :ie antibody. The levels of bacteria can be detected by measuring the < -new I a i Fluor Cray fiat is emitted.
EXAMPLES
Example 1 Real time PCR to measure total plaque bacteria levels 100281 Levels of total plaque bacteria (micrograms bacterial DNA r1) in subjects is measured using different toothpaste formulations, using the procedures described supra:
Total bacterial DNA S. mutans DNA S. sanguis DNA
250 ppm fluoride 6.091 0.09622 1.126 formulation (control) 1450 ppm fluoride 6.018 0.09903 1.1.0 7 formulation Formulation having 3.781 0.05998 1.291 21V O arginine bicarbonate and 1450 ppm fluoride 100291 The arginine-fluoride formulation is effective to reduce total bacterial plaque loads, and S. rnutans (cariogenic) plaque loads, while enhancing S. sanguis (arginolytic) loads.
Example 2 -- Ammonia production (00301 Ammonia production is measured in subjects using different toothpaste forrtulatios, using the method described above:
Ammonia level (ppm) 250 ppm fluoride 1.97 formulation (control) 1450 ppm fluoride 1.79 I- _-,r; is i n having 2.77 r,e w:id 14450 1 or.1e 100311 A monia production is significantly higher in plaque of subjects using the arginine-containing formulation.
Example 3 Lactic acid levels 100321 Plaque lactic acid levels are measured in subjects using capillary electrophoresis as described above, showing that lactate is significantly increased in the presence of sucrose.
Sucrose Plaque Challenged Plaque Lactate 1.87 7.82 0.37 nmollmg) Example 4 - Real time PCR ! RT-PCR
100331 This invention combines the principles of real-time PCR detection of bacterial species with the use of messenger RNA (mRNA) as an indicator of biological activity within cells. Following purification of mRNA from a bacterial sample, reverse transcription real time PCR is used to detect and quantify specific bacteria within a simple or complex environment. The invention covers the sequence of the primers as well as the mRNA
identification method and its application.
100341 One function of DNA within viable cells is to code for the synthesis of proteins.
DNA codes for its corresponding mRNA strand which is then used as the instructions for assembling finished proteins. Unlike DNA, mRNA has a very short half life (seconds to minutes) and is only present in cells that are either viable or very recently killed. Whereas DNA is present in cells in a fixed number of copies, mRNA levels are often changed. in response to the conditions in which a cell exists. Expression. of different proteins may be up-or downregulated in response to temperature changes, growth media, growth phases and other environmental conditions. Therefore, ifthe target gene is not carefully chosen, it is possible that fluctuations in env-ironmrental condiE=on, ~%.:11 be falsely rà as population a the ' L cc i a~ y u d : t. c ' because ;ei # à r .et sequence- Th, 3 sequence ha as a Tl . n ',I tuf expression has been observed under different i n ntal conditions.
[00351 Real time PCR uses the basic chemistry behind polynaeras~ reaction (.PC'R) amplification of genetic material and couples it with real time detection k ,C
i1 zorescent labels as a mechanism of quantifying the number of copies of a given genetic sequence pr :c nt after each amplification cycle. The simplest of these methods uses SYBR Green I, a fluorescent probe that intercalates specifically into double stranded DNA (dsDNA).
Increasing levels of SYBR Green fluorescence therefore correlate to greater concentrations of dsDN
A. When this dye is included in a PCR reaction primed using specific genetic sequences, the increase in fluorescence corresponds to an increase in the number of copies of the target gene.
Subsequently, the cycle number at which the signal crosses a predetermined intensity threshold can be correlated to the concentration of the genetic sequence in the starting material.
100361 The development of real time PCR technology has made it possible to detect and quantify specific biological species rapidly and with a high degree of accuracy. Conventional methods for quantification of bacterial species rely on the development of primers to the variable region of the DNA encoding the 16s ribosomal subunit. This subunit is critical to bacterial replication and its sequence is, therefore, not readily mutated. The detection of 6s rDNA sequences specific to a particular species can facilitate the detection and enumeration of a single bacterial species within a complex environment.
100371 Primers are designed based on the sequences of wuf genes from publically available databases (National Center for Biotechnology Information. and the Los Alamos Oral Pathogens Database). Sequences are aligned using the DNA Star Lasergene program MegAlign module. This alignment is used to select a region of greater divergence in order to maximize the likelihood of species specificity. Primer sequences are selected based upon analysis information available from the Roche Diagnostics LightCycler Probe Design software. Primers covered by this invention include not only those already designed and tested, but all primers to this genetic region in oral pathogens.
100381 Total RNA is isolated from samples using an appropriate RNA isolation kit or other RNA isolation method. Any preferred method for RNA isolation can be used. Purified RNA is treated 2 times with appropriate DNase treatment reagents. This step degrades any DN' :R\; :)rep and `l l i I,I i i, },<i: is )sitives.
t. } 4 11s. T'he re .r` ) ieH: and 1 .
c,.-mr ol for the conl ,,-'C.Le Civic a real P01' reverse transcription step. PCK Jr,'i uct, obtained. in the abcLi, c of .1 reverse Iran reaction must be the result of acing DNA_ I
100391 A standard curve is generated by performing the real time reverse transcription PCR reaction on RN.A samples isolated from cultures containing known amounts of viable bacteria. The second derivative maximum value for each known sample is plotted against its known concentration of bacterial cells. The second derivative maximum of amplification curves of RNA isolated from unknown bacterial samples can then be compared to the standard curve to determine the concentration of viable organisms within the sample population. This data would be valuable information in deterring the effects of antibacterials and active molecules on the ecology of the oral environment.
100401 The following primer pair is designed to amplify a 228 base pair region of the trtf-gene from AggregatibactÃ:r (9.ctinohacilia s) actinomyceetemcomitans:
Primer Sequence T. ( C) %GC AG Annealing name (kcal.nlol) Temp.
Forward 5' AAGCGCGTGG rATCAC 3' 49.05 56.25 -27.56 55 C
Reverse 5' -- TG'TAAGGAACACCTA .-.._..-. 3' 31.52 40.00 -20.15 55 C
Table 1. Properties of primers designed for quantification of mRNA expression of tufin A.
actinomvicet (: Inc om itans .
100411 These primers are used to amplify RNA isolated from both pure cultures of A.
actinrnoyceterncomitans and mixed populations both with and without A.
aetinomviceterncomitans included. The results, in particular the relationship between fluorescence (Fl) and cycle number are shown in the graph of Figure 1. In Figure 1, "water"
represents the negative control and "Aa" is the positive control of pure A.
actinontycetetncomitans culture. "Mix 1" was purified from a population containing Prevo/tella interniedia, Streptococcus sobrinus, Streptococcus oralis, and Actinornyces tiiscosus and should, therefore be negative for amplification with these primers. "Mix 3" is from a population containing A. acctino tyc.,etemcÃ3nnitans, Por 3hyrom 3nas gingiralis, trervo oc cus cr, ~? C ??r7Zl. Streptococcus t? itans, and t rrptoc'occus sanquinrs and should be M0421 This I'-is ampl. f c i "'i; i .-:: 1.:r' curve o liicJ positive s r true iiu rn !..
,cJcing ii :.3lit}'Jv~ g ;t.' ::ITIi.:`.
amp are able to aL.
detect r 4.:'trl;: 3a J tan from within m 'x of RNV%
100431 The ability of these primers to accurately detect and quantify only viable .ac t$rarrrr~r c~c~t rr~t'omitrrrts organisms is determined as follows. A
known concentration of A.
aÃctinrormzycetWrrracornitcars cells are killed by suspending in 80% ethanol for 15 minutes. The bacteria are then pelleted by centrifugation and resuspended in fresh Brain Heart Infusion broth growth media. The ethanol killed bacteria are incubated overnight at 37 C and examined for growth to confirm that no viable organisms were remained. The ethanol killed bacteria are then mixed, in defined ratios, with viable organisms and reverse transcription followed by real time PCR is performed. The amplification of these samples is shown in Figure 2, which shows the amplification of RNA from mixes of live and dead A_ aetinomyeetenicornltan.s.
100441 Despite the fact that all of the populations used as templates for this reaction contained the same total number of organisms, earlier amplification is observed in samples containing more viable organisms, indicating that this assay is able to detect viable organisms within a mix of both live and dead bacteria. Additionally, the melting curve of these samples, as shown in Figure 3, indicates that a single, identical product is amplified in all samples, which demonstrates the high specificity of the assay. Figure 3 shows a melting peak analysis of products amplified from pure and mixed cultures of .1.
aetinomycetemcornitans. The overlap of these curves indicates that a single product is being amplified from all samples.
Table 2 shows a comparison of expected and calculated number of organisms in selected standard curve samples.
Sample Expected viable CFU Calculated viable CFU
100% 5.00 x 10 5.34 x 10' 50% 2.50 x 10' 2.28 x 10' 40% 2.00 x 107 2.54 x 10 30% 1.50x 10' 1.18x107 1.00 x 10 1.07 x 10' 5.00 x 106' 4 06 x 1 2.50x 10 .ic x T able 2. t c_, ud and caicLuated number ..x, _. ;: 'n selected standard Lars i 100451 Lased on the known concentrations of t le , . ,~,nd killed starting cultures, the approximate number of viable organisms in each popuiation is calculated and used in.
Ix conjunction with the second derivative maximum of each amplification curve to generate a standard curve. The results are shown in Figure 4, which illustrates a standard curve and linear regression of the standard curve generated from the amplification of known concentrations of viable and dead A. actimm L L ('-~~ .L.==rnitans. The r2 value of the regression line is 0.96.
100461 The r' value of a linear regression line indicates the closeness of fit of the regression equation to the observed values. An r2 closer to 1.00 indicates that the observed values match closely to the regression line. For the example above, the r-value of the standard curve is 0.96, indicating that about 96% of the total variation observed in the line is due to actual measured variation in the samples and that this standard curve can be used to calculate the concentration of viable organisms in unknown populations.
O0471 In practice, in a single experiment, the concentration of viable organisms calculated based on this standard curve is not significantly different from the actual concentration added prior to RNA isolation and differed by <20%. These data indicate that this assay represents a rapid, accurate means of detecting and quantifying viable organisms of a specific species within a complex population of organisms. This represents a potentially powerful tool for analyzing the effects of treatments on oral microbial ecology.
Claims (12)
1. A method to assess the bioflora of the oral cavity comprising measuring levels of arginolytic bacteria.
2. The method of claim 1wherein cariogenic bacterial activity is also measured.
3. The method of claim 1 or 2 wherein the arginolytic bacterial activity is assessed by measuring plaque ammonia production levels.
4. The method of claim 2 or 3 wherein the cariogenic bacterial activity is assessed by measuring lactate production levels.
5. A method of any of the foregoing claims comprising using quantitative real time PCR, quantitative RT-PCR, and/or fluorescent antibody probes to quantify levels of at least one cariogenic bacteria and/or at least one arginolytic bacteria.
6. A method according to any of the foregoing claims wherein the arginolytic bacteria includes S. sanguis.
7. A method according to any of the foregoing claims wherein cariogenic bacterial activity is also measured and the cariogenic bacteria includes S. mutans.
8. A method of any of the foregoing claims wherein the method detects potentially damaging changes in plaque ecology before there is measurable or significant demineralization or damage to the teeth.
9. A method to enhance oral health, e.g., to a. reduce or inhibit formation of dental caries, b. reduce or inhibit demineralization and promote remineralization of the teeth, c. treat, reduce or inhibit formation of early enamel lesions.
d. reduce hypersensitivity of the teeth, e. reduce or inhibit gingivitis, f. promote healing of sores or cuts in the mouth, g. reduce levels of acid producing bacteria, h. increase relative levels of arginolytic bacteria, i. inhibit microbial biofilm formation in the oral cavity.
j. raise and/or maintain plaque pH at levels of at least pH 5.5 following sugar challenge, k. reduce plaque accumulation, l. treat, relieve or reduce dry mouth, in. whiten teeth, n. enhance systemic health, including cardiovascular health, o. immunize or protect teeth against cariogenic bacteria, p. clean the teeth and oral cavity and/or q. reduce erosion of the teeth comprising measuring the bioflora of the oral cavity using a method according to any of the foregoing claims, and if indicated, by the presence of elevated levels of cariogenic bacteria and/or elevated lactate levels, and/or the presence of low levels of arginolytic bacteria and/or low levels of plaque ammonia production, administering an oral care product comprising an effective amount of a basic amino acid or salt thereof.
d. reduce hypersensitivity of the teeth, e. reduce or inhibit gingivitis, f. promote healing of sores or cuts in the mouth, g. reduce levels of acid producing bacteria, h. increase relative levels of arginolytic bacteria, i. inhibit microbial biofilm formation in the oral cavity.
j. raise and/or maintain plaque pH at levels of at least pH 5.5 following sugar challenge, k. reduce plaque accumulation, l. treat, relieve or reduce dry mouth, in. whiten teeth, n. enhance systemic health, including cardiovascular health, o. immunize or protect teeth against cariogenic bacteria, p. clean the teeth and oral cavity and/or q. reduce erosion of the teeth comprising measuring the bioflora of the oral cavity using a method according to any of the foregoing claims, and if indicated, by the presence of elevated levels of cariogenic bacteria and/or elevated lactate levels, and/or the presence of low levels of arginolytic bacteria and/or low levels of plaque ammonia production, administering an oral care product comprising an effective amount of a basic amino acid or salt thereof.
10. The method of claim 9 wherein the basic amino acid is arginine or salt thereof.
11. Use of a basic amino acid, in free or salt form, for the manufacture of medicament for enhancing oral health in a subject whose oral cavity bioflora comprise elevated levels of cariogenic bacteria and/or elevated lactate levels, and/or low levels of arginolytic bacteria and/or low levels of plaque ammonia production, as measured by a method according to any of the foregoing claims.
12. A method for cosmetically enhancing the oral cavity which method comprises claims 1 to 10, and if indicated by the presence of elevated levels of cariogenic bacteria and/or low levels of plaque ammonia production, administering an oral care product comprising a basic amino acid in free or salt form.
Applications Claiming Priority (11)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US2742008P | 2008-02-08 | 2008-02-08 | |
| US2743208P | 2008-02-08 | 2008-02-08 | |
| US2743108P | 2008-02-08 | 2008-02-08 | |
| US61/027,431 | 2008-02-08 | ||
| US61/027,432 | 2008-02-08 | ||
| US61/027,420 | 2008-02-08 | ||
| US2743708P | 2008-02-09 | 2008-02-09 | |
| US2744208P | 2008-02-09 | 2008-02-09 | |
| US61/027,437 | 2008-02-09 | ||
| US61/027,442 | 2008-02-09 | ||
| PCT/US2009/033287 WO2009100262A2 (en) | 2008-02-08 | 2009-02-06 | Oral care methods and systems |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2711265A1 true CA2711265A1 (en) | 2009-08-13 |
Family
ID=40952691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2711265A Abandoned CA2711265A1 (en) | 2008-02-08 | 2009-02-06 | Assessment of oral cavity bioflora |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US9376722B2 (en) |
| EP (1) | EP2225562B1 (en) |
| JP (2) | JP2011510683A (en) |
| CN (1) | CN102037360B (en) |
| AU (1) | AU2009212318B2 (en) |
| CA (1) | CA2711265A1 (en) |
| CO (1) | CO6300814A2 (en) |
| MX (1) | MX2010005211A (en) |
| MY (1) | MY155568A (en) |
| RU (1) | RU2476890C2 (en) |
| WO (1) | WO2009100262A2 (en) |
| ZA (1) | ZA201003680B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2476890C2 (en) | 2008-02-08 | 2013-02-27 | Колгейт-Палмолив Компани | Oral care methods and systems |
| US9649265B2 (en) | 2010-12-09 | 2017-05-16 | Colgate-Palmolive Company | Liquid cleaning composition containing long-chain fatty acid |
| RU2567052C2 (en) | 2010-12-09 | 2015-10-27 | Колгейт-Палмолив Компани | Liquid cleaning compositions, containing long-chain fatty alcohols |
| CN103348240A (en) | 2011-02-11 | 2013-10-09 | 高露洁-棕榄公司 | Methods of analyzing plaque |
| JP2016509025A (en) | 2013-02-14 | 2016-03-24 | ユニバーシティ オブ フロリダ リサーチ ファンデーション インコーポレーティッド | Probiotic arginine degradable oral composition and method for producing and using probiotic arginine degradable oral composition |
| US10206959B2 (en) | 2013-02-14 | 2019-02-19 | University Of Florida Research Foundation, Inc. | Probiotic arginolytic oral compositions and methods of making and using probiotic arginolytic oral compositions |
| CN104721063B (en) | 2013-12-19 | 2018-05-08 | 高露洁-棕榄公司 | Dentrifice composition comprising zinc oxide and zinc citrate |
| RU2673473C2 (en) * | 2013-12-27 | 2018-11-27 | Колгейт-Палмолив Компани | Prebiotic oral care compositions containing carboxylic acids |
| CA3053680A1 (en) * | 2017-03-03 | 2018-09-07 | The Procter & Gamble Company | Calcium fluorescent probes to assess oral care composition efficacy in biofilm |
| BR112021011686A2 (en) * | 2018-12-26 | 2021-09-08 | Colgate-Palmolive Company | PREBIOTIC ROLE OF ARGININE IN BENEFICIAL ORAL BACTERIA |
| BR112021011600A2 (en) * | 2018-12-26 | 2021-08-31 | Colgate-Palmolive Company | METHODS OF SWITCHING BIOFILM IN THE ORAL CAVITY FROM PATHOGENIC TO HEALTHY BIOFILM |
Family Cites Families (94)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3538230A (en) * | 1966-12-05 | 1970-11-03 | Lever Brothers Ltd | Oral compositions containing silica xerogels as cleaning and polishing agents |
| US3678154A (en) | 1968-07-01 | 1972-07-18 | Procter & Gamble | Oral compositions for calculus retardation |
| US3535421A (en) | 1968-07-11 | 1970-10-20 | Procter & Gamble | Oral compositions for calculus retardation |
| US4152418A (en) * | 1970-04-01 | 1979-05-01 | Lever Brothers Company | Zinc and enzyme mouthwash and mouthwash concentrate for reducing dental plaque and calculus formation |
| US3696191A (en) * | 1970-11-10 | 1972-10-03 | Monsanto Co | Dental creams containing enzymes |
| US3943241A (en) | 1971-08-30 | 1976-03-09 | General Mills, Inc. | Cariostatic composition |
| US3932608A (en) | 1971-08-30 | 1976-01-13 | General Mills, Inc. | Food composition |
| US4058595A (en) * | 1971-10-13 | 1977-11-15 | Colgate-Palmolive Company | Stabilized toothpastes containing an enzyme |
| US3932605A (en) | 1972-06-12 | 1976-01-13 | Jaroslav Vit | Dental treatment |
| US3988434A (en) | 1972-08-07 | 1976-10-26 | Schole Murray L | Dental preparation |
| US3959458A (en) * | 1973-02-09 | 1976-05-25 | The Procter & Gamble Company | Oral compositions for calculus retardation |
| US4025616A (en) | 1973-03-06 | 1977-05-24 | The Procter & Gamble Company | Oral compositions for plaque, caries and calculus retardation with reduced staining tendencies |
| US3937807A (en) * | 1973-03-06 | 1976-02-10 | The Procter & Gamble Company | Oral compositions for plaque, caries, and calculus retardation with reduced staining tendencies |
| US3862307A (en) * | 1973-04-09 | 1975-01-21 | Procter & Gamble | Dentifrices containing a cationic therapeutic agent and improved silica abrasive |
| US4100269A (en) | 1973-06-28 | 1978-07-11 | Lever Brothers Company | Anticalculus dentifrice |
| US4022880A (en) | 1973-09-26 | 1977-05-10 | Lever Brothers Company | Anticalculus composition |
| US3925543A (en) | 1973-11-01 | 1975-12-09 | Colgate Palmolive Co | Antibacterial oral compositions containing preservative-antioxidants |
| US3991177A (en) * | 1973-11-27 | 1976-11-09 | Colgate-Palmolive Company | Oral compositions containing dextranase |
| CA1063357A (en) * | 1974-05-21 | 1979-10-02 | James J. Benedict | Abrasive composition |
| US4011309A (en) | 1975-01-20 | 1977-03-08 | Marion Laboratories, Inc. | Dentifrice composition and method for desensitizing sensitive teeth |
| US4051234A (en) * | 1975-06-06 | 1977-09-27 | The Procter & Gamble Company | Oral compositions for plaque, caries, and calculus retardation with reduced staining tendencies |
| US4064138A (en) | 1975-11-12 | 1977-12-20 | General Mills, Inc. | Amino acid derivatives |
| ZA773318B (en) | 1976-06-18 | 1978-04-26 | I Kleinberg | Means and method for improving natural defenses against caries |
| USRE31181E (en) | 1976-06-18 | 1983-03-15 | Means and method for improving natural defenses against caries | |
| US4108981A (en) | 1976-08-02 | 1978-08-22 | Indiana University Foundation | Alkaline oral compositions comprising aluminum and a carboxylic acid |
| US4042680A (en) | 1976-08-02 | 1977-08-16 | Indiana University Foundation | Anticariogenic maloaluminate complexes |
| US4108979A (en) | 1976-08-02 | 1978-08-22 | Indiana University Foundation | Dentifrice preparations comprising aluminum and a compatible abrasive |
| US4146607A (en) | 1977-11-07 | 1979-03-27 | Lever Brothers Company | Synergistic anti-plaque mixture with tetradecylamine plus aluminum and/or zinc |
| US4160821A (en) | 1978-02-27 | 1979-07-10 | Johnson & Johnson | Treatment for gingivitis |
| US4213961A (en) | 1978-03-23 | 1980-07-22 | Beecham, Inc. | Oral compositions |
| GB1573727A (en) | 1978-05-19 | 1980-08-28 | Colgate Palmolive Co | Dentifrices |
| US4225579A (en) | 1979-02-27 | 1980-09-30 | Israel Kleinberg | Means and method for improving defenses against caries |
| US4340583A (en) * | 1979-05-23 | 1982-07-20 | J. M. Huber Corporation | High fluoride compatibility dentifrice abrasives and compositions |
| US4339432A (en) | 1979-06-20 | 1982-07-13 | Lever Brothers Company | Oral mouthwash containing zinc and glycine |
| US4269822A (en) | 1979-07-20 | 1981-05-26 | Laclede Professional Products, Inc. | Antiseptic dentifrice |
| JPS5846483B2 (en) | 1979-09-20 | 1983-10-17 | ライオン株式会社 | Oral composition |
| JPS5835965B2 (en) | 1979-07-31 | 1983-08-05 | ライオン株式会社 | Oral composition |
| US4355022A (en) * | 1981-07-01 | 1982-10-19 | Interon, Inc. | Method of dental treatment |
| US4532124A (en) | 1981-08-19 | 1985-07-30 | Development Finance Corporation Of New Zealand | Dental rinse |
| JPS58118509A (en) | 1981-12-29 | 1983-07-14 | Lion Corp | Composition for oral cavity |
| US4885155A (en) * | 1982-06-22 | 1989-12-05 | The Procter & Gamble Company | Anticalculus compositions using pyrophosphate salt |
| US4725576A (en) | 1983-12-29 | 1988-02-16 | Research Foundation Of State University Of New York | Fungicidal polypeptide compositions containing L-histidine and methods for use therefore |
| US4528181A (en) | 1984-02-01 | 1985-07-09 | Colgate-Palmolive Company | Dentifrice containing dual sources of fluoride |
| US5334617A (en) | 1984-03-19 | 1994-08-02 | The Rockefeller University | Amino acids useful as inhibitors of the advanced glycosylation of proteins |
| GB8411731D0 (en) | 1984-05-09 | 1984-06-13 | Unilever Plc | Oral compositions |
| US5000939A (en) * | 1984-06-12 | 1991-03-19 | Colgate-Palmolive Company | Dentifrice containing stabilized enzyme |
| JPH0742219B2 (en) | 1984-07-26 | 1995-05-10 | ライオン株式会社 | Oral composition |
| US4538990A (en) | 1984-09-24 | 1985-09-03 | Medical College Of Ga. Research Institute, Inc. | Method of decreasing the permeability of a dental cavity |
| US4976951A (en) * | 1985-08-28 | 1990-12-11 | Melvyn Rosenberg | Dental caries diagnostic and localization technique |
| US5043154A (en) * | 1987-01-30 | 1991-08-27 | Colgate-Palmolive Co. | Antibacterial, antiplaque, anticalculus oral composition |
| US5032386A (en) * | 1988-12-29 | 1991-07-16 | Colgate-Palmolive Company | Antiplaque antibacterial oral composition |
| CH671879A5 (en) * | 1987-02-26 | 1989-10-13 | Nestle Sa | |
| US4866161A (en) * | 1987-08-24 | 1989-09-12 | University Of South Alabama | Inhibition of tartar deposition by polyanionic/hydrophobic peptides and derivatives thereof which have a clustered block copolymer structure |
| US5004597A (en) * | 1987-09-14 | 1991-04-02 | The Procter & Gamble Company | Oral compositions comprising stannous flouride and stannous gluconate |
| GB8729564D0 (en) | 1987-12-18 | 1988-02-03 | Unilever Plc | Oral compositions |
| US4842847A (en) * | 1987-12-21 | 1989-06-27 | The B. F. Goodrich Company | Dental calculus inhibiting compositions |
| US5438076A (en) * | 1988-05-03 | 1995-08-01 | Perio Products, Ltd. | Liquid polymer composition, and method of use |
| US5334375A (en) | 1988-12-29 | 1994-08-02 | Colgate Palmolive Company | Antibacterial antiplaque oral composition |
| US4954137A (en) * | 1989-12-19 | 1990-09-04 | Shell Oil Company | Inhibition of sulfide inclusion in slag |
| US5096700A (en) | 1990-09-28 | 1992-03-17 | The Procter & Gamble Company | Halogenated aminohexanoates and aminobutyrates antimicrobial agents |
| US5370865A (en) | 1992-05-15 | 1994-12-06 | Kao Corporation | Composition for use in oral cavity |
| US5286480A (en) | 1992-06-29 | 1994-02-15 | The Procter & Gamble Company | Use of N-acetylated amino acid complexes in oral care compositions |
| DE4306673A1 (en) * | 1993-03-04 | 1994-09-08 | Hoechst Ag | Process for the preparation of a dicalcium phosphate dihydrate suitable for use in toothpastes |
| JP3566374B2 (en) | 1994-02-03 | 2004-09-15 | 花王株式会社 | Oral composition |
| JP3803695B2 (en) | 1994-11-28 | 2006-08-02 | サンスター株式会社 | Antibacterial preparation |
| WO1997002802A1 (en) | 1995-07-10 | 1997-01-30 | Unilever N.V. | Self-heating dentifrice |
| US5762911A (en) | 1996-03-05 | 1998-06-09 | The Research Foundation Of State University Of New York | Anti-caries oral compositions |
| US6488961B1 (en) | 1996-09-20 | 2002-12-03 | Ethypharm, Inc. | Effervescent granules and methods for their preparation |
| US5906811A (en) | 1997-06-27 | 1999-05-25 | Thione International, Inc. | Intra-oral antioxidant preparations |
| US5922346A (en) | 1997-12-01 | 1999-07-13 | Thione International, Inc. | Antioxidant preparation |
| US6850883B1 (en) * | 1998-02-09 | 2005-02-01 | Nokia Networks Oy | Decoding method, speech coding processing unit and a network element |
| UA72206C2 (en) * | 1998-03-04 | 2005-02-15 | Dandy S | Coated chewing gum, method of obtaining thereof and using one or several active ingredients in solid form |
| US6805883B2 (en) | 1998-03-12 | 2004-10-19 | Mars, Incorporated | Food products containing polyphenol(s) and L-arginine to stimulate nitric oxide |
| US5997301A (en) | 1998-10-20 | 1999-12-07 | Linden; Lars Ake | Treatment of tooth surfaces and substances therefor |
| US6436370B1 (en) * | 1999-06-23 | 2002-08-20 | The Research Foundation Of State University Of New York | Dental anti-hypersensitivity composition and method |
| US6558654B2 (en) * | 2000-04-11 | 2003-05-06 | Mclaughlin Gerald | Composition and method for whitening teeth |
| AUPQ909000A0 (en) * | 2000-07-28 | 2000-08-24 | University Of Sydney, The | A method of detecting microorganisms |
| US6876806B2 (en) | 2000-11-14 | 2005-04-05 | Optiva, Inc. | Optical waveguides and method of fabrication thereof |
| US20020081360A1 (en) | 2000-12-27 | 2002-06-27 | Andreas Burgard | Salts of L-amino acid having improved taste and their preparation |
| DE10108900B4 (en) * | 2001-02-23 | 2005-03-10 | 3M Espe Ag | Determination of patient-related caries risk |
| JP4268358B2 (en) * | 2001-12-18 | 2009-05-27 | 株式会社トクヤマデンタル | Antibody and immunological assay |
| AU2003301404A1 (en) * | 2002-10-15 | 2004-05-04 | The Regents Of The University Of California | Monoclonal antibodies specific for cariogenic bacteria |
| CA2516950C (en) * | 2003-02-21 | 2012-09-25 | Rhodia Inc. | Anti-sensitivity, anti-caries, anti-staining, anti-plaque, ultra-mild oral hygiene agent |
| US20040258630A1 (en) * | 2003-06-23 | 2004-12-23 | Boyd Thomas J. | Antiplaque breath freshening consumable film |
| GB0323211D0 (en) | 2003-10-03 | 2003-11-05 | Toshiba Res Europ Ltd | Signal decoding methods and apparatus |
| MX348545B (en) | 2005-07-12 | 2017-06-16 | Colgate-Palmolive Company * | Oral care implement having reservoir for dispensing active agent. |
| US20070014740A1 (en) | 2005-07-15 | 2007-01-18 | Colgate-Palmolive Company | Oral compositions having cationic active ingredients |
| GB0525369D0 (en) | 2005-12-14 | 2006-01-18 | Ineos Silicas Ltd | Silicas |
| RU2308036C1 (en) | 2006-04-18 | 2007-10-10 | Государственное образовательное учреждение высшего профессионального образования "Тверская государственная медицинская академия Федерального агентства по здравоохранению и социальному развитию" (ГОУ ВПО Тверская ГМА Росздрава) | Method of individual estimation of sensitivity of mouth microflora to antimicrobial agents |
| RU2476890C2 (en) | 2008-02-08 | 2013-02-27 | Колгейт-Палмолив Компани | Oral care methods and systems |
| RU2543656C2 (en) | 2008-02-08 | 2015-03-10 | Колгейт-Палмолив Компани | Oral care product and method of using and making it |
| PA8774301A1 (en) | 2008-02-08 | 2009-09-17 | Colgate Palmolive Co | ORAL CARE REGIME |
| US20090202456A1 (en) | 2008-02-08 | 2009-08-13 | Colgate-Palmolive Company | Novel salts and their uses |
| JP2011510982A (en) | 2008-02-08 | 2011-04-07 | コルゲート・パーモリブ・カンパニー | Oral care products and methods of use and manufacture thereof |
-
2009
- 2009-02-06 RU RU2010137332/15A patent/RU2476890C2/en active
- 2009-02-06 MY MYPI2010002284A patent/MY155568A/en unknown
- 2009-02-06 CN CN200980104889.5A patent/CN102037360B/en not_active Expired - Fee Related
- 2009-02-06 AU AU2009212318A patent/AU2009212318B2/en not_active Ceased
- 2009-02-06 MX MX2010005211A patent/MX2010005211A/en unknown
- 2009-02-06 CA CA2711265A patent/CA2711265A1/en not_active Abandoned
- 2009-02-06 EP EP09708171.5A patent/EP2225562B1/en active Active
- 2009-02-06 WO PCT/US2009/033287 patent/WO2009100262A2/en active Application Filing
- 2009-02-06 JP JP2010546013A patent/JP2011510683A/en active Pending
- 2009-02-06 US US12/866,614 patent/US9376722B2/en active Active
-
2010
- 2010-05-24 ZA ZA2010/03680A patent/ZA201003680B/en unknown
- 2010-08-31 CO CO10107446A patent/CO6300814A2/en not_active Application Discontinuation
-
2014
- 2014-01-06 JP JP2014000302A patent/JP2014138586A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JP2014138586A (en) | 2014-07-31 |
| EP2225562B1 (en) | 2019-06-26 |
| CN102037360B (en) | 2016-10-05 |
| EP2225562A4 (en) | 2016-04-27 |
| JP2011510683A (en) | 2011-04-07 |
| ZA201003680B (en) | 2015-08-26 |
| CN102037360A (en) | 2011-04-27 |
| WO2009100262A2 (en) | 2009-08-13 |
| RU2476890C2 (en) | 2013-02-27 |
| EP2225562A2 (en) | 2010-09-08 |
| MY155568A (en) | 2015-10-30 |
| RU2010137332A (en) | 2012-03-20 |
| WO2009100262A3 (en) | 2009-11-05 |
| AU2009212318B2 (en) | 2012-12-13 |
| US20110085990A1 (en) | 2011-04-14 |
| AU2009212318A1 (en) | 2009-08-13 |
| US9376722B2 (en) | 2016-06-28 |
| CO6300814A2 (en) | 2011-07-21 |
| MX2010005211A (en) | 2010-09-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2009212318B2 (en) | Oral care methods and systems | |
| Suzuki et al. | Quantitative analysis of multi-species oral biofilms by TaqMan Real-Time PCR | |
| Stingu et al. | Microbial profile of patients with periodontitis compared with healthy subjects. | |
| Khalichi et al. | The influence of triethylene glycol derived from dental composite resins on the regulation of Streptococcus mutans gene expression | |
| Furukawa et al. | Highly sensitive real-time PCR assay for quantification of toxic cyanobacteria based on microcystin synthetase A gene | |
| JP2020141686A (en) | Oral examination method | |
| Dame-Teixeira et al. | Presence of Archaea in dental caries biofilms | |
| Salvador et al. | Evaluating the influence of light intensity in mcyA gene expression and microcystin production in toxic strains of Planktothrix agardhii and Microcystis aeruginosa | |
| Gillis et al. | Characterisation of short tandem repeats for genotyping Mycobacterium leprae | |
| JP2008206516A (en) | Method for measuring number of oral streptococci, and pcr primer and probe set used for the same | |
| Matondkar et al. | Quantitative assessment of Scardovia wiggsiae from dental plaque samples of children suffering from severe early childhood caries and caries free children | |
| Singer et al. | Quantitative assessment of urea, glucose and ammonia changes in human dental plaque and saliva following rinsing with urea and glucose | |
| JP4917815B2 (en) | Method for detecting periodontal disease bacteria | |
| JP4878186B2 (en) | Method for detecting periodontal disease bacteria | |
| JP6579874B2 (en) | Oligonucleotide set for periodontal pathogen detection and method for detecting periodontal pathogen | |
| TWI424063B (en) | Oral care methods and systems | |
| JP4359699B2 (en) | Method for detecting carious bacteria and primer set used in the method | |
| EP1975257B1 (en) | A method for measuring the number of oral lactobacillus, and a PCR primers-probe set used for the same | |
| Geevarghese et al. | Application of 16s RRNA in identifying oral microflora-a review of literature | |
| RU2796603C1 (en) | Set of reagents for the detection of streptococcus mutans by isothermal loop amplification | |
| RU2788617C1 (en) | Test system for early detection of porphyromonas gingivalis involved in inflammatory reactions | |
| EP0550610B1 (en) | Processes, oligonucleotide primers and oligonucleotide probes for the detection of pathogenic bacteria in the buccal cavity | |
| CN101983245B (en) | Kit and method for determining whether or not unmethylated cytosine conversion treatment is appropriately performed and method for analyzing methylated DNA using the same | |
| Horz et al. | Current molecular technologies for assessing the amount of microbial pathogens in oral plaque biofilms | |
| KR20220075628A (en) | Method of providing information for diagnosis of dental caries and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |
Effective date: 20160708 |