阅读说明：本技术 一种口腔类微生物数量快速测定方法、试剂盒及应用 (Method and kit for rapidly determining number of oral microorganisms and application of kit ) 是由 崔大祥 李雪玲 梁辉 田静 吕军鸿 于 2021-09-01 设计创作，主要内容包括：本发明公开了一种口腔类微生物数量快速测定方法、试剂盒及应用,本发明所述测定方法包括下列步骤：(1)将待测样品进行ATP裂解释放；(2)在上述混合液中加入荧光检测液,混合均匀；(3)用荧光检测仪读取荧光值；(4)根据ATP标准品制作的标准曲线,获得微生物数量。本发明方法灵敏度高、成本低廉、操作简易,能够快速有效地测定口腔类微生物数量,可用于口腔清洁类产品抑菌效果评价和临床诊断等领域。(The invention discloses a rapid determination method of the number of oral microorganisms, a kit and application thereof, wherein the determination method comprises the following steps: (1) carrying out ATP cracking release on a sample to be detected; (2) adding a fluorescence detection solution into the mixed solution, and uniformly mixing; (3) reading the fluorescence value by using a fluorescence detector; (4) the microbial count was obtained according to a standard curve prepared from an ATP standard. The method has the advantages of high sensitivity, low cost and simple operation, can quickly and effectively determine the number of the oral microorganisms, and can be used in the fields of antibacterial effect evaluation and clinical diagnosis of oral cleaning products and the like.)

1. A method for rapidly measuring the number of oral microorganisms adopts an ATP standard substance to make a standard curve, and is characterized by comprising the following steps:

(1) carrying out ATP cracking release on a sample to be detected;

(2) adding a fluorescence detection solution into the mixed solution, and uniformly mixing;

(3) reading the fluorescence value by using a fluorescence detector;

(4) the microbial count was obtained according to a standard curve prepared from an ATP standard.

2. The method for rapidly measuring the quantity of the oral microorganisms according to claim 1, wherein the sample to be measured comprises clinical diagnosis samples and pure cultures of the oral microorganisms, wherein the clinical diagnosis samples comprise one or more of swabs and saliva.

3. The method for rapid determination of the number of microorganisms in the oral cavity according to claim 1, wherein the ATP lysis release method comprises one of surfactant lysis release, heated boiling lysis release, ultrasonic lysis release, acid lysis release or base lysis release, wherein the surfactant comprises one or more of cetyltrimethylammonium bromide, benzalkonium chloride, dihydroxythreitol, sodium dodecyl sulfate, trichloroacetic acid, and the like.

4. The method of claim 1, wherein the fluorescence detection solution comprises 32mg luciferase, 25.2mg D-luciferin, and 112mg bovine serum albumin per 60mL ATP fluorogenic reagent buffer solution, wherein the ATP fluorogenic reagent buffer solution comprises 4.468g Tris, 0.732g disodium EDTA, 3.232g magnesium acetate, 26.8mg dithiothreitol, 100g beta-cyclodextrin, and 3.7g glucose dissolved in 1000mL water.

5. The method for rapidly measuring the number of microorganisms in the oral cavity according to claim 1, wherein the fluorescence detector comprises one or more of a biochemical luminescence detector, a real-time fluorescence detector and a portable fluorescence detector.

6. The method for rapidly measuring the number of microorganisms in the oral cavity according to claim 1, wherein the ATP standard curve is prepared according to the following steps:

1) adding 0.5mmol/L ATP standard into 400 μ L ice-bath sterile physiological saline, gently vibrating, and mixing to obtain 10^{- 4}Diluting the ATP standard substance by mol/L; then, the user can use the device to perform the operation,

2) sequentially diluting by 10 times to obtain 10^-5～10^-10A standard substance serial dilution of mol/L;

3) lightly vibrating and mixing the ATP standard dilution with 0.03% CTAB according to the volume ratio of 10:1 respectively, and 4^oC centrifuge, 10000gCentrifuging for 5 minutes, and performing ATP lysis release;

adding 20 mu L of gradient ATP standard dilution into 100 mu L of self-prepared fluorescence detection solution, uniformly mixing, and reacting for 3 minutes;

reading the fluorescence value by using a fluorescence detector;

and sequentially detecting three parallel samples by each dilution gradient, and calculating an average value, wherein a logarithm value of the concentration of the ATP standard substance is used as an abscissa, and a logarithm value of the corresponding fluorescence intensity is used as an ordinate, so as to draw a standard curve of the ATP standard substance.

7. The method for rapidly measuring the number of microorganisms in the oral cavity according to any one of claims 1 to 6, wherein the rapid measurement of the number of Staphylococcus aureus, which is an oral bacterium, comprises the steps of:

1) drawing a standard curve of the content and the fluorescence intensity of each gradient colony of the staphylococcus aureus, and carrying out the following steps:

culturing a purchased staphylococcus aureus strain on a nutrient broth slope for 20 hours, flushing the slope with 10ml of sterile normal saline, and uniformly mixing the staphylococcus aureus strain with the sterile normal saline to obtain a bacterial suspension;

the bacterial suspension is added in 4^oCentrifuge C, 4000rpm, centrifuge for 8 minutes, wash with sterile physiological salineThe precipitate was precipitated several times, and then, the precipitate was suspended in 10ml of physiological saline;

taking 1ml of bacterial suspension, and performing 10-fold gradient dilution to 10 by using sterile physiological saline^-8(ii) a Taking the last 5 gradient bacterial suspensions to count the plate bacterial colonies according to a national standard method;

respectively taking each gradient bacterium suspension and 0.03 percent CTAB according to the volume ratio of 10:1, oscillating and mixing uniformly, carrying out ice bath ultrasonic treatment for 10 minutes, and then 4^oC centrifuge, 11000gCentrifuging for 5 minutes, uniformly mixing 20 mu L of supernate with 100 mu L of fluorescence detection solution, and reacting for 3 minutes;

reading the fluorescence value by using a fluorescence detector;

taking the logarithm value of the microbial quantity obtained by a flat plate counting method as an abscissa and taking the logarithm value of the corresponding fluorescence intensity as an ordinate, and drawing a standard curve of the content of each gradient bacterial colony of the microbe and the fluorescence intensity;

obtaining a unitary linear equation through regression analysis, and showing that the logarithm of the concentration of each colony and the logarithm of the corresponding fluorescence value are in positive correlation;

2) and calculating the corresponding microorganism content at a certain luminescence value by the standard curve or the unary linear equation.

8. The method for rapidly measuring the number of the oral microorganisms according to claim 7, wherein the staphylococcus aureus is replaced by other oral microorganisms such as escherichia coli or staphylococcus epidermidis, and a standard curve of the colony content and the fluorescence intensity of each gradient is drawn to obtain a univariate linear equation.

9. A kit for use in a method for rapid determination of the number of microorganisms in the oral cavity as claimed in any one of claims 1 to 8, wherein the kit comprises one or more of a surfactant, luciferin, luciferase, ATP standard.

10. Use of the method for rapidly determining the number of microorganisms in the oral cavity according to any one of claims 1 to 8, for evaluating the bacteriostatic effect of an oral cleaning product.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly determining the number of oral microorganisms, a kit and application.

Background

A large number of microorganisms are propagated in the human mouth, which may include those that induce dental caries, gingivitis and periodontitis or stomatitis. Therefore, the measurement of the number of the oral microorganisms has great significance for the diagnosis and prognosis of oral diseases, the evaluation of the bacteriostatic effect of oral cleaning products and the like. The existing method for measuring the number of oral microorganisms mainly comprises microorganism plate culture, and although the method has high sensitivity and is a 'gold standard' for measuring the number of microorganisms, the method has the defects of complex operation, long required time, incapability of measuring the number of microorganisms which cannot be cultured and the like. Therefore, it is an urgent need to solve the problem for those skilled in the art to find a rapid determination method for the number of oral microorganisms, which is not dependent on culture, and has simple operation and high sensitivity.

The ATP bioluminescence detection is designed based on a luminescence mechanism of a bioluminescence system, a special luminescent substance-luciferin and luciferase are arranged in a bioluminescent cell, and the luciferin is activated by Adenosine Triphosphate (ATP) to emit fluorescence photons under the catalysis of the luciferase. Since all living cells of an organism contain a constant amount of ATP, the ATP content clearly indicates how much of the microorganisms and other organisms remain in the sample. The fluorescence intensity and ATP concentration are in linear relation in a certain range. The ATP released by the microorganisms can be measured by using the luminophor system, so that the content of the microorganisms can be measured, and a simple and sensitive method is provided for detecting the microorganisms within minutes. At present, no report that the ATP bioluminescence detection method is used for measuring the number of microorganisms in the oral cavity exists.

Disclosure of Invention

The invention aims to provide a method for rapidly measuring the number of oral microorganisms. So as to overcome the problems of complex operation, long time, incapability of measuring the number of the culturable microorganisms and the like in the current oral microorganism counting method.

Yet another object of the present invention is to: provides a kit for rapidly determining the number of oral microorganisms.

Yet another object of the present invention is to: the application of the method for rapidly determining the number of the oral microorganisms is the antibacterial effect evaluation of the oral cleaning products.

The purpose of the invention is realized by the following scheme: a method for rapidly determining the number of oral microorganisms, which is characterized by comprising the following steps:

(1) carrying out ATP cracking release on a sample to be detected;

(2) adding a fluorescence detection solution into the mixed solution, and uniformly mixing;

(3) reading the fluorescence value by using a fluorescence detector;

(4) the microbial count was obtained according to a standard curve prepared from an ATP standard.

The invention overcomes the problems of complex operation, long time, incapability of measuring the number of the culturable microorganisms and the like in the conventional oral microorganism counting method. Based on the characteristics of the oral microorganisms, the method for measuring the number of the oral microorganisms provided by the ATP bioluminescence detection method has the advantages of simple operation, short detection time and high sensitivity, and reduces the omission factor of the microorganisms which cannot be cultured.

The sample to be detected comprises one or more of clinical diagnosis samples and pure cultures of oral microorganisms. The clinical diagnosis sample comprises one or more of swabs and saliva.

The ATP cracking and releasing method comprises one of cracking and releasing methods of surfactant cracking and releasing, heating and boiling cracking and releasing, ultrasonic cracking and releasing, acid cracking and releasing, alkali cracking and releasing and the like.

The fluorescence detection solution comprises one or more of luciferin and luciferase. The fluorescence detection solution contains 32mg of luciferase, 25.2mg of D-luciferin and 112mg of bovine serum albumin per 60mL of ATP fluorescent reagent buffer solution. Wherein, the ATP fluorescent reagent buffer solution is prepared by dissolving 4.468g of tris (hydroxymethyl) aminomethane, 0.732g of disodium ethylene diamine tetraacetate, 3.232g of magnesium acetate, 26.8mg of dimercaptothreitol, 100g of beta-cyclodextrin and 3.7g of glucose in each 1000mL of water.

The fluorescence detector comprises a biochemical luminescence detector, a real-time fluorescence detector, a portable fluorescence detector and the like.

The invention provides a kit for rapidly determining the number of oral microorganisms, which is characterized by comprising one or more of a surfactant, luciferin, luciferase and an ATP standard substance.

The surfactant is characterized by comprising one or more of Cetyl Trimethyl Ammonium Bromide (CTAB), benzalkonium bromide (BAB), benzalkonium chloride (BAC), Dihydroxythreitol (DTT), Sodium Dodecyl Sulfate (SDS), trichloroacetic acid (TCA) and the like.

The invention provides application of a method for rapidly determining the number of oral microorganisms, which is characterized in that the application is antibacterial effect evaluation of an oral cleaning product.

The ATP standard product is used for drawing a standard curve. Under the condition of consistent with the experimental conditions of the ATP standard, the drawing of the standard curve is unnecessary when the sample to be tested is subsequently tested.

The invention provides a rapid determination method for the number of oral microorganisms in the fields of oral cleaning products, clinical diagnosis and the like. The beneficial effects of the invention include: the method for determining the number of the oral microorganisms has the advantages of convenience in operation, high detection speed, high sensitivity and the like. Compared with the conventional microbial culture method, the ATP-based bioluminescence detection principle is adopted, microbial culture is not required, and complicated steps of preparation of a culture medium, plate coating, colony counting and the like are avoided, so that the method is very suitable for antibacterial effect evaluation and clinical diagnosis of oral cleaning products. Any combination of the above preferred conditions is within the scope of the present invention based on the general knowledge in the art.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples, but the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1

The method for drawing the standard curve of the ATP standard substance comprises the following steps:

adding ATP standard (0.5 mmol/L) into ATP standard, adding 400 μ L ice-bath sterile physiological saline, vibrating gently, and mixing to obtain 10^-4Diluting ATP standard substance by mol/L, and sequentially diluting by 10 times to obtain 10^-5～10^-10A standard substance serial dilution of mol/L; mixing ATP standard sample with 0.03% CTAB at volume ratio of 10:1, and mixing 4^oC centrifuge, 10000gCentrifuging for 5 minutes, and performing ATP lysis release;

adding 20 mu L of gradient ATP diluent into 100 mu L of self-prepared fluorescence detection solution, uniformly mixing, and reacting for 3 minutes; the fluorescence detection solution comprises 32mg of luciferase, 25.2mg of D-luciferin and 112mg of bovine serum albumin in every 60mL of ATP fluorogenic reagent buffer solution, wherein 4.468g of tris (hydroxymethyl) aminomethane, 0.732g of disodium edetate, 3.232g of magnesium acetate, 26.8mg of dimercaptothreitol, 100g of beta-cyclodextrin and 3.7g of glucose are dissolved in every 1000mL of water;

reading the fluorescence value by using a fluorescence detector;

and sequentially detecting three parallel samples by each dilution gradient, and calculating an average value, wherein a logarithm value of the concentration of the ATP standard substance is used as an abscissa, and a logarithm value of the corresponding fluorescence intensity is used as an ordinate, so as to draw a standard curve of the ATP standard substance.

By regression analysis, a unary linear equation of y =0.8546x +9.843 (R)²=0.9965，P<0.05), indicating that the standard curve prepared from the ATP standard is representative.

Example 2

The method for rapidly measuring the number of the oral bacteria comprises the following steps:

1, staphylococcus aureus is an important pathogenic bacterium of multi-gap infection of oral cavity, jaw and face, and the staphylococcus aureus strain used in the experiment of the embodiment is purchased from China center for culture and Collection of Industrial microorganisms (CICC) with the serial number of 21600;

culturing staphylococcus aureus on the slant of the nutrient broth for 20 hours, flushing the slant with 10ml of sterile normal saline, and uniformly mixing the washed staphylococcus aureus with the sterile normal saline to obtain a bacterial suspension;

the bacterial suspension is added in 4^oC, centrifuging for 8 minutes at 4000rpm by using a centrifuge, and washing the precipitate for several times by using sterile physiological saline; then, the precipitate was suspended in 10ml of physiological saline;

taking 1ml of the bacterial suspension stock solution, and performing 10-fold gradient dilution to 10 by using sterile physiological saline^-8；

Taking the last 5 gradient bacterial suspensions to count the plate bacterial colonies according to a national standard method;

respectively taking each gradient bacterium suspension and 0.03 percent CTAB according to the volume ratio of 10:1, oscillating and mixing uniformly, carrying out ultrasonic treatment in ice bath for 10 minutes, and then 4^oC centrifuge, 11000gCentrifuging for 5 minutes, and taking supernatant;

20 mu L of the supernatant and 100 mu L of the self-prepared fluorescence detection solution (the preparation steps are consistent with those of the example 1) are uniformly mixed and reacted for 3 minutes;

reading the fluorescence value by using a fluorescence detector;

taking the logarithm value of the microbial quantity obtained by a flat plate counting method as an abscissa and taking the logarithm value of the corresponding fluorescence intensity as an ordinate, and drawing a standard curve of the content of each gradient bacterial colony of the microbe and the fluorescence intensity;

by regression analysis, a unary linear equation of y =0.9926x-1.843 (R)²=0.9432，P<0.05), indicating that the log concentration of each colony is positively correlated with the log of the corresponding fluorescence value.

The corresponding microorganism content at a certain luminescence value can be calculated by the standard curve or the unary linear equation.

According to the method, standard curves of the content and the fluorescence intensity of each gradient colony of the Escherichia coli (the strain is purchased from China center for Industrial culture Collection of microorganisms, number 10421) and the Staphylococcus epidermidis (the strain is purchased from China center for general culture Collection of microorganisms, number 1.4260) are respectively drawn to obtain a unary linear equation.

The results of analysis of variance of the colony contents of the microorganisms under different fluorescence values show that the colony concentration differences corresponding to several common microorganisms are not significant, namely, the influence of strain differences can be ignored when the ATP fluorescence counting is used for measuring the number of the microorganisms.

Application example

The application of the rapid determination method of the number of the oral bacteria in the antibacterial effect evaluation of the oral cleaning products is as follows:

kits were prepared according to the reagent components used in example 1 and example 2; the oral cleaning product is gargle G prepared by the company.

Dividing the saliva into three parts, wherein the first part is placed at room temperature for 3 minutes; the second part and clean water are mixed evenly for 3 minutes in equal volume proportion; mixing the third part and gargle G at equal volume ratio for 3 min; then, the user can use the device to perform the operation,

according to the method of example 2, two swabs were subjected to lysis release of ATP using CTAB in the kit; then, the user can use the device to perform the operation,

adding a fluorescence detection solution; subsequently, the process of the present invention,

the number of microorganisms was measured in a portable fluorescence detector (wind path ATP fluorescence detector).

The results show that the microbial contents in the three saliva samples are 3000, 1500 and 48 respectively, which shows that the mouth cleaning product gargle G has good antibacterial effect.

The gingival surface was gently scraped 3 times with swab a, then rinsed 3 minutes with mouthwash G, and then gently scraped 3 times with another swab B at the same location. Then, according to the method of example 2, two swabs were subjected to lysis release of ATP using CTAB in the kit, and then a fluorescence detection solution was added, followed by measurement of microbial numbers in a portable fluorescence detector (aeolian ATP fluorescence detector).

The results show that the numbers of the microorganisms contained in the swab A and the swab B are 180 and 5 respectively, and the oral cleaning product mouthwash G has a good antibacterial effect.

The results show that the rapid determination method for the number of the oral bacteria can be well used for evaluating the antibacterial effect of the oral cleaning products.