TWI424063B - Oral care methods and systems - Google Patents

Description

口腔保健方法及系統Oral health care method and system

本申請案主張2008年2月9日申請之美國專利申請案序號61/027,437案之優先權；亦請求2008年2月9日申請之美國專利申請案序號61/027,442案、及均於2008年2月8日申請之美國專利申請案序號61/027,432、61/027,431、61/027,420、與61/027,435等案之優先權；該等申請案之內容全部併入本文以資參考。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.

本發明係有關使用含有呈游離或鹽型鹼性胺基酸之組成物測量口中致齲齒及分解精胺酸細菌之相對量之方法(例如，為牙齒保健法之一部分)。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).

精胺酸及其他鹼性胺基酸已被建議用於口腔保健及被認為在對抗蛀牙形成及牙齒敏感上具有重大益處。市售可得之精胺酸系牙膏為含CaviStat之DenClude與ProClude(其中含有精胺酸與碳酸氫鈣)。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).

口中生物菌叢種類通常於形成蛀牙及口腔衛生上扮演重要角色。舉例而言，一般假設精胺酸有利作用之重要因素在於其與其他鹼性胺基酸可被特定種類細菌[例如血鏈球菌(S .sanguis )]代謝，彼等細菌不會產生齲齒且與致齲齒細菌[例如變形鏈球菌(S .mutans )]競爭牙齒上與口腔中之位置。分解精胺酸之細菌可利用精胺酸及其他鹼性胺基酸產生氨，因而提高其環境之pH值；而致齲齒細菌代謝糖則產生乳酸，傾向於降低牙菌斑之pH及去除牙齒之礦物質，最後導致蛀牙。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.

若有有效方法可監測口中生物菌叢之種類以，例如，決定最適療法及監測病患治療之有效性，將會很有用。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.

本發明提供用於評估口中生物菌叢之迅速又簡單之方法。The present invention provides a rapid and simple method for assessing bacterial flora in the mouth.

於第一具體實例中，本發明測定牙菌斑氨產生量以確定分解精胺酸細菌之相對族群。In a first embodiment, the invention measures plaque ammonia production to determine the relative population of bacteria decomposing arginine.

於另一具體實例中，本發明測定牙菌斑乳酸量以確定致齲齒細菌之相對族群。In another embodiment, the invention determines the amount of plaque lactic acid to determine the relative population of cariogenic bacteria.

於另一具體實例中，本發明使用聚合酶鏈反應(PCR)(例如定量即時PCR)以鑑定口中(例如，牙菌斑或唾液中)之生物菌叢。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).

於另一實例中，本發明使用反轉錄酶PCR(RT-PCR)以鑑定口中(例如，牙菌斑或唾液中)之生物菌叢。In another example, the invention uses reverse transcriptase PCR (RT-PCR) to identify a bacterial flora in the mouth (eg, in plaque or saliva).

於另一具體實例中，係使用抗體探針(例如，螢光抗體探針)以鑑定口中(例如，牙菌斑或唾液中)之生物菌叢。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).

舉例而言，本發明定量至少一種致齲齒細菌(例如，變形鏈球菌)及至少一種分解精胺酸細菌(例如，血鏈球菌)之量。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).

於另一具體實例中，係使用前述方法之一者評估病患，並據此指示治療。In another embodiment, the patient is assessed using one of the methods described above and the treatment is indicated accordingly.

本發明方法特別係用於檢測牙菌斑生態之潛在有害變化，而於對牙齒有可測量或顯著脫礦化或傷害之前得以進行矯正治療。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.

本發明因而提供增進口腔健康之方法以，例如The invention thus provides a method of improving oral health, for example

a.減少或抑制形成齲齒；a. reducing or inhibiting the formation of dental caries;

b.減少或抑制牙齒之脫礦化作用及促進礦化作用；b. reducing or inhibiting demineralization of teeth and promoting mineralization;

c.治療、減少或抑制早期牙釉質病變；c. treating, reducing or inhibiting early enamel lesions;

d.減少牙齒之過敏反應；d. reduce the allergic reaction of the teeth;

e.減少或抑制牙齦炎；e. reduce or inhibit gingivitis;

f.促進口中潰瘍或傷口之癒合；f. promote the healing of ulcers or wounds in the mouth;

g.減少產酸細菌量；g. reduce the amount of acid-producing bacteria;

h.增加分解精胺酸細菌之相對量；h. increase the relative amount of bacteria decomposing arginine;

i.抑制口腔中微生物生物膜之形成；i. inhibiting the formation of microbial biofilms in the oral cavity;

j.於糖刺激後提升及/或維持牙菌斑pH於至少pH 5.5之程度；j. increasing and/or maintaining the plaque pH to at least pH 5.5 after stimulation with sugar;

k.減少牙菌斑堆積；k. reduce plaque accumulation;

l.治療、緩和或減少口乾；l. Treat, alleviate or reduce dry mouth;

m.美白牙齒；m. whitening teeth;

n.增進全身健康，包括心血管健康(例如，利用減少經由口腔組織之全身性感染之可能性)；n. Promote general health, including cardiovascular health (eg, the use of reduced likelihood of systemic infection through oral tissues);

o.使牙齒對致齲齒細菌及其作用免疫；o. Immunize the teeth against caries bacteria and their effects;

p.清潔牙齒及口腔及/或p. cleaning teeth and mouth and / or

q.減少牙齒腐蝕；q. Reduce tooth corrosion;

該方法包括測量口腔之生物菌叢(例如，使用前述任何方法)，若必要，則投與含有效量鹼性胺基酸(例如，精胺酸)或其鹽之口腔保健產品。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.

本發明進一步提供使用呈游離或鹽型之鹼性胺基酸製造藥劑，針對根據本發明方法所測量，其口腔生物菌叢含有致齲齒細菌量上升及/或乳酸量上升、及/或分解精胺酸細菌量及/或低牙菌斑氨產生量低之患者，增進口腔健康之用途。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.

本發明進一步提供增進口腔美觀之方法(其中該等增進美觀可包括例如使牙齒更潔白及/或減少口臭)，該方法包括使用根據本發明之方法測量口腔生物菌叢，若指示出現致齲齒細菌量升高及/或乳酸量升高、及/或出現分解精胺酸細菌量下降及/或牙菌斑氨產生量下降，則投與含有呈游離或鹽型鹼性胺基酸之口腔保健產品。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.

牙菌斑代謝-氨產生量Plaque metabolism - ammonia production

牙菌斑轉化精胺酸為氨之能力係分解精胺酸活性之指標。某些細菌具有轉化精胺酸為氨之能力，正如同某些細菌會轉化糖類為酸。增加分解精胺酸菌株之相對濃度是有利的，因為彼等細菌產生對致齲齒細菌增殖(此增殖偏愛酸性條件及增加蛀牙風險)不利之條件。每天使用精胺酸被預期可以類似經常攝食糖產生有利於產酸細菌的條件之方式而產生有利於分解精胺酸細菌之牙菌斑生態轉移。氨為能中和酸之鹼，有助於維持中性牙菌斑pH。中性pH條件對非病原細菌較為有利。對照於測定所挑選之分解精胺酸細菌之濃度而未區分具代謝活性(活)與失活(死)細菌之即時PCR法(於下文進一步敘述)，測量氨產生量可測定能轉化精胺酸為氨的所有細菌之貢獻。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.

氨檢測套組為市售可得，例如，得自Diagnostic Chemicals Limited(Oxford,CT)，可測定氨產生量，其定量及測定之原理為已知氨與α-酮戊二酸及還原型菸鹼醯胺腺嘌呤二核苷酸磷酸(NADPH)反應形成L-麩胺酸及NADP；此反應由麩胺酸脫氫酶(GLDH)催化；由於NADPH氧化而於340nm之吸光率降低係與氨濃度成正比。經預定治療流程後，收集牙菌斑試樣。於若干應用中，牙菌斑係自裝於固定器(retainer)上之釉質或HAP樣品獲得。於其他應用中，牙菌斑直接自牙齒獲取。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.

根據乳酸量之牙菌斑生態Plaque ecology based on the amount of lactic acid

正如同測量氨量作為衡量分解精胺酸細菌量之代理物(proxy)，乳酸係衡量致齲齒細菌量之代理物。進行牙菌斑取樣之患者從前一天晚上即不再進食或喝水，早上也不刷牙；以10%蔗糖溶液沖洗2分鐘；8分鐘後，刮除牙齒表面，收集牙菌斑。將牙菌斑試樣收集於置於冰上之預先稱重之管中，並測量牙菌斑重。其分析包括添加冰冷的水於已知量牙菌斑試樣中，接著加熱試樣至80℃ 5分鐘以殺死細菌及釋出所有酸，然後將試樣置於冰水中冷卻5分鐘。接著離心試樣，過濾上澄液。使用毛細管電泳法測定乳酸濃度。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.

根據定量即時PCR之牙菌斑生態Plaque ecology based on quantitative real-time PCR

定量即時PCR(聚合酶鏈反應)係定量DNA之高度靈敏方法。由於DNA量與存在之細菌量直接相關，因此使用單離自牙菌斑之細菌DNA定量總細菌量。即時PCR被例如美國疾病防治中心(Center for Disease Control)與FDA等政府機構公認為強有力且靈敏之技術。利用許多口腔細菌之已知基因體序列，設計探針以檢測口腔細菌或特定細菌例如變形鏈球菌或血鏈球菌之總量。利用聚合酶鏈反應擴增單離自牙菌斑或唾液試樣之DNA。DNA量隨各PCR反應循環呈指數遞增。由於反應係經使用螢光報導分子後即時進行，因此該技術被稱為“即時”。於本發明之一具體實例中，係使用SYBR Green作為報導分子。此分子與雙股DNA配合後強烈發螢光。利用設定螢光臨界值(threshold)及使用各種濃度之DNA標準確定達到該臨界值需要之循環數而達成定量。存在的DNA愈多，達到臨界值需要之DNA循環數愈少。市售之即時PCR儀器可自許多廠商(例如Roche Diagnostics)購得。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.

牙菌斑試樣係自具已知及固定表面積之牙釉質或羥基磷灰石樣本獲得。牙菌斑收集之標準化相當具關鍵性，因為DNA存在量與收集之牙菌斑量直接關聯。使用牙菌斑量作為即時PCR測定的總細菌量標準化之方法並不適當，因為此二量顯著相關。結果記錄為每毫升之微克DNA。可就DNA濃度或Ln(DNA濃度)進行統計分析。關於總細菌量(total bacteria)係使用患者及治療為因子進行二因子變異數分析(ANOVA)。若差異性檢測出95%信賴標準，則彼等差異性被視為顯著。就特定細菌例如變形鏈球菌或血鏈球菌而言，係使用總細菌量作為互變量進行二因子變異數分析。特定細菌總量由於與細菌族群總量有關，因此為牙菌斑生態健康更恰當之指標。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.

於本發明之特定具體實例中，測量變形鏈球菌以作為致齲齒性之指標；選用變形鏈球菌乃因其為與啟動蛀牙有關之公認危險因子。雖有其他產酸細菌涉及蛀牙過程，惟一般已知變形鏈球菌特別在致齲過程開始及早期扮演重要角色。於本發明一具體實例中，選用血鏈球菌作為轉移至更健康牙菌斑生態之指標；因一般已知血鏈球菌為具高分解精胺酸活性(轉化精胺酸為氨之能力)之細菌。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.

根據RT-PCR之牙菌斑生態Plaque ecology according to RT-PCR

反轉錄PCR測定試樣中之RNA轉錄本。單離出RNA，使用反轉錄酶將轉錄本轉化為cDNA，並使用PCR擴增cDNA。RT-PCR之優點為DNA系之口腔細菌檢測法無法檢測彼等菌種之存活性。因為口腔細菌最常於生物膜群落中被發現，死細菌之DNA可於死後長時期保留於生物膜結構中。其他方法，例如螢光系存活性測定法(Live Dead kit,Molecular Probes)，可檢測生物體是否具有受損害之膜(compromised membranes)，惟不能直接檢測特定菌種。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.

因此反轉錄即時PCR方法為定量存在複雜群落中之特定口腔細菌菌株活生物體之方法。mRNA具相當短之半衰期，因此為新近活細菌之指標。申請人等已開發出對延伸因子tuf (elongation factortuf )具菌種特異性之引子。此基因不受生長階段、培養基或環境條件明顯調控，因而將所檢測細菌數之虛擬影響減至最小。使用放線共生放線桿菌(Aggregatibacter actinomycetemcomitans )作為測試生物體，於少至20%活生物體存在時，亦可於活細菌與被EtOH殺死的細菌之混合族群中檢測出存活差異性。此外，此方法得以於含有多達六種不同細菌菌種之混合菌種族群中可靠地鑑定放線共生放線桿菌之存在。所計算之細菌濃度與根據相同培養物之OD₆₁₀ 估計之數值密切相關(r=0.96，差異性<1%)。此測定法代表於口腔複雜環境中研究特定生物體生態之方法。於有進一步之基因序列資料可利用時，即可開發出寬廣種類口腔細菌之引子。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.

根據螢光抗體探針之細菌量Bacterial amount based on fluorescent antibody probe

使用蛀牙診斷套組，經由使用單株抗體以檢測唾液中之致齲齒型細菌(例如，變形鏈球菌)及/或非致齲齒型細菌(例如，血鏈球菌)之量。所用特定抗體對細菌菌種具特異性及具有附加於抗體之螢光。細菌量可利用測量發射之螢光量予以檢測。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.

實例Instance 實例1Example 1 測量牙菌斑細菌總量之即時PCRReal-time PCR for measuring the total amount of plaque bacteria

患者之牙菌斑細菌總量(微克細菌DNA/毫升)係使用各種牙膏調配劑，以前文所述程序進行測量：The total amount of plaque bacteria in the patient (microgram bacterial DNA/ml) was measured using various toothpaste formulations, as described previously:

精胺酸-氟化物調配劑有效地減少總細菌牙菌斑荷載量與變形鏈球菌(致齲齒)牙菌斑荷載量，而增加血鏈球菌(分解精胺酸)荷載量。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).

實例2-氨產生量Example 2 - Ammonia production

氨產生量係使用各種牙膏調配劑，以上文所述程序進行測量：Ammonia production is measured using a variety of toothpaste formulations, as described above:

使用含精胺酸調配劑患者牙菌斑中之氨產生量明顯較高。The amount of ammonia produced in plaque of patients using arginine-containing formula was significantly higher.

實例3-乳酸量Example 3 - Amount of lactic acid

患者牙菌斑乳酸量係如上文所述使用毛細管電泳法進行測量，結果顯示於蔗糖存在下，乳酸顯著增加。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.

實例4-即時PCR/RT-PCRExample 4 - Real-time PCR / RT-PCR

本發明結合使用信使RNA(mRNA)作為細胞內生物活性指標進行細菌菌種即時PCR檢測之原理。自細菌試樣純化mRNA後，使用反轉錄即時PCR檢測及定量單純或複雜環境中之特定細菌。本發明涵蓋諸引子之序列以及mRNA鑑定方法及其應用。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.

活細胞中DNA之一功能為編碼以合成蛋白質。DNA編碼其對應之mRNA股，接著被作為組裝最終蛋白質之指南。與DNA不同，mRNA具有很短之半衰期(數秒至數分鐘)，且只存在活或剛死亡之細胞中；DNA以固定套數存在細胞中，而mRNA量則常反應細胞存活之條件而變化。不同蛋白質之表現可能反應溫度變化、生長培養基、生長階段與其他環境條件而向上或向下調節。因此，若未小心挑選標靶基因，則環境條件之變動很可能被錯誤解讀為係族群存活性之變動。為了避免彼等效應，本發明使用延伸因子tu，其基因tuf ，作為標靶序列。因為在不同實驗條件下，tuf 表現甚少變化或無變化，因此此序列先前已被作為標記使用。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.

即時PCR使用支持遺傳物質聚合酶鏈反應(PCR)擴增作用之基礎化學，並連接螢光標記之即時檢測(其為定量各擴增循環後存在之既定基因序列套數之機制)。彼等方法之最簡易者為使用SYBR Green I，一種特異性地嵌入雙股DNA(dsDNA)中之螢光探針；因此SYBR Green螢光增加量與較高dsDNA濃度相關。當此染料併入主要使用特定基因序列之PCR反應中時，則螢光之增加相當於標靶基因套數之增加。隨後，訊號越過預定強度臨界值之循環數與起始物質中基因序列之濃度相關。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.

即時PCR技術之開發已使其可迅速且高精確度地檢測及定量特定生物物種。用於定量細菌菌種之習知方法依賴針對編碼16s核糖體次單元DNA可變區之引子之開發。此次單元對細菌複製具關鍵性，因此，其序列不易突變。對特定菌種具特異性之16s rDNA序列之檢測有助於複雜環境中單一細菌菌種之檢測與計數。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.

引子之設計係根據得自公開可用資料庫(National Center for Biotechnology Information and the Los Alamos Oral Pathogens Database)之諸tuf 基因之序列。使用DNA Star Lasergene程式MegAlign標準元件排列諸序列。此排列係用於挑選具較大分岐性之區域，俾使菌種特異性之相似度增至最大。引子序列之挑選係根據得自Roche Diagnostics LightCycler Probe Design軟體之資訊分析。本發明所涵蓋之引子不僅包括已經設計及測試者，亦包括針對口腔病原菌此基因部位之所有引子。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.

使用適當RNA單離套組或其他RNA單離方法自試樣中單離全部RNA；可使用單離RNA之任何較佳方法。經純化之RNA以適當DNase處理試劑處理2次；此步驟降解RNA製備物內任何污染之DNA，及防止獲得偽陽性結果。然後反轉錄單離之RNA，以產生互補DNA(cDNA)分子；擴增所得cDNA，使用SYBR Green進行檢測。可進行無反轉錄步驟之即時PCR反應，以作為完全去除DNA之品質管控；缺乏反轉錄反應獲得之PCR產物必為污染DNA之結果。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.

針對自含已知量活細菌之培養物單離之RNA試樣進行即時反轉錄PCR反應，以產生標準曲線。以各已知試樣之二階導數(second derivative)最大值對其細菌細胞之已知濃度作圖。接著可將自未知細菌試樣單離的RNA擴增曲線之二階導數最大值與該標準曲線進行比較，以確定試樣族群中活生物體之濃度。此數據於遏制抗細菌劑與活性分子對口腔環境生態之影響時將為有價之資訊。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.

下述引子對係設計用以擴增得自放線共生放線桿菌tuf 基因之一228個鹼基對區域：The following primer pairs were designed to amplify a 228 base pair region derived from the Tuf gene of Actinobacillus symbiosis:

彼等引子係用於擴增自放線共生放線桿菌之純培養物及包含或不含放線共生放線桿菌之混合族群二者單離之RNA；其結果，特別是螢光(F1)與循環數間之關係，示於圖1。於圖1中，“water”代表純放線共生放線桿菌培養物之負對照組及“Aa”為其正對照組；“Mix 1”係自含中間普雷沃菌(Prevoltella intermedia )、遠緣鏈球菌(Streptococcus sobrinus )、口腔鏈球菌(Streptococcus oralis )、與黏放線菌(Actinomyces viscosus )之族群純化，因此使用彼等引子之擴增應為陰性；“Mix 3”係得自含放線共生放線桿菌、牙齦卟啉單胞菌(Porphyromonas gingivalis )、格氏鏈球菌(Streptococcus gordonii )、變形鏈球菌、與血鏈球菌之族群，放線共生放線桿菌之擴增應為陽性。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.

此圖表證明，在含放線共生放線桿菌之混合族群以與正對照組類似之曲線進行擴增之同時，缺乏放線共生放線桿菌之混合族群則遵循與水對照組相同之擴增曲線；表示彼等引子能自各種RNA混合物中精確地檢測出放線共生放線桿菌。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.

彼等引子僅精確地檢測及定量活放線共生放線桿菌生物體之能力可如下文所述確定。便已知濃度之放線共生放線桿菌細胞懸浮於80%乙醇15分鐘進行殺菌。然後離心使細菌沉澱，沉澱物再懸浮於新鮮Brain Heart Infusion液體生長培養基中。於37℃培養此經乙醇殺死之細菌隔夜，檢查其生長以證實無活生物體存留。然後使經乙醇殺死之細菌以既定比率與活生物體混合，進行反轉錄及隨後之即時PCR。彼等試樣之擴增示於圖2，其中顯示得自活及死放線共生放線桿菌混合族群之RNA之擴增。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.

儘管作為此反應模板用之所有族群含有相同總量之生物體，惟於含較多活生物體之試樣中觀察到較早之擴增，表示此測定法能於活及死細菌混合族群中檢測出活生物體。此外，彼等試樣之熔融曲線，如圖3所示，顯示所有試樣中有單一、相同產物被擴增，證明該測定法之高度特異性。圖3顯示自純放線共生放線桿菌及混合培養物擴增之產物之熔融峰分析。彼等曲線重疊表示所有試樣中有單一產物被擴增。表2顯示於選定標準曲線試樣中生物數預測值與計算值之比較。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.

根據活及被殺死之起始培養物之已知濃度，計算各族群中活生物體之大略數量，與各擴增曲線之二階導數最大值結合使用，以產生標準曲線；其結果如圖4所示，其說明自已知濃度活及死放線共生放線桿菌之擴增產生之標準曲線及該標準曲線之線性迴歸；迴歸線之r² 值為0.96。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.

線性迴歸線之r² 值表示迴歸等式配合觀測值之接近性。r² 接近1.00表示觀測值與迴歸線密切符合。關於上述實例，標準曲線之r² 值為0.96，表示此線中觀測之約96%總變異係由於試樣中實際測量之變異，因而此標準曲線可用於計算未知族群中活生物體之濃度。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.

實際上，於單一實驗中，根據此標準曲線計算出之活生物之濃度與進行RNA單離前所添加之實際濃度並無顯著差異，此差異<20%。彼等數據表示，此測定法代表於複雜生物族群中檢測及定量特定菌株活生物體之迅速、精確之方法；係用於分析諸項處理對口腔微生物生態的效應之潛在有力工具。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.

圖1　得自純培養物及混合菌種族群之放線共生放線桿菌之擴增。Figure 1 Amplification of Actinobacillus actinomycetemii from pure cultures and mixed bacterial populations.

圖2　得自活及死放線共生放線桿菌混合族群之RNA之擴增。Figure 2 Amplification of RNA from a mixed population of live and dead line symbiosis A. faecalis.

圖3　自純放線共生放線桿菌及其混合培養物擴增之產物之熔融峰分析。Figure 3 is a melting peak analysis of products amplified from pure line A. faecalis and its mixed cultures.

圖4　自已知濃度活及死放線共生放線桿菌之擴增產生之標準曲線及該標準曲線之線性迴歸。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)

一種評估口腔生物菌叢之方法，該方法包括測量分解精胺酸細菌之量及測量致齲齒細菌之量，其中該分解精胺酸細菌之量係利用測量牙菌斑氨產生量進行評估，及該致齲齒細菌之量係利用測量乳酸產生量進行評估，且其中該分解精胺酸細菌之量及該致齲齒細菌之量係使用定量即時PCR、定量RT-PCR、及/或螢光抗體探針以定量。 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. 根據申請專利範圍第1項之方法，其中該分解精胺酸細菌包括血鏈球菌(S.sanguis )。The method of claim 1, wherein the lysine-degrading bacterium comprises S. sanguis . 根據申請專利範圍第1項之方法，其中該致齲齒細菌包括變形鏈球菌(S.mutans )。The method of claim 1, wherein the cariogenic bacteria comprises S. mutans . 如申請專利範圍第1項之方法，其中該方法係於對牙齒有可測量或顯著脫礦化或傷害之前檢測牙菌斑生態之潛在有害變化。 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. 一種鹼性胺基酸或其鹽用於製造口腔保健產品之用途，該產品係用於在由根據申請專利範圍第1至4項中任一項之方法測量之口腔生物菌叢包含致齲齒細菌量上升及/或乳酸量上升、及/或分解精胺酸細菌量下降及/或牙菌斑氨產生量下降之個體中：a.減少或抑制形成齲齒；b.減少或抑制牙齒之脫礦化作用及促進礦化作用；c.治療、減少或抑制早期牙釉質病變； d.減少牙齒之過敏反應；e.減少或抑制牙齦炎；f.促進口中潰瘍或傷口之癒合；g.減少產酸細菌量；h.增加分解精胺酸細菌之相對量；i.抑制口腔中微生物生物膜之形成；j.於糖刺激後提升及/或維持牙菌斑pH於至少pH 5.5之程度；k.減少牙菌斑堆積；l.治療、緩和或減少口乾；m.美白牙齒；n.增進全身健康，包括心血管健康；o.保護牙齒對抗致齲齒細菌或對其免疫；p.清潔牙齒及口腔及/或q.減少牙齒腐蝕。 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. 如申請專利範圍第5項之用途，其中該鹼性胺基酸為精胺酸或其鹽。 The use of the fifth aspect of the invention, wherein the basic amino acid is arginine or a salt thereof. 一種鹼性胺基酸或其鹽用於製造口腔保健產品之用途，該產品係在由根據申請專利範圍第1至4項中任一項之方法測量之口腔生物菌叢包含致齲齒細菌量上升及/或乳酸量上升、及/或分解精胺酸細菌量下降及/或牙菌斑氨產生量下降之個體中增進口腔美觀。 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.