Reagent for caries activity test and use method
A reagent using resazurin sodium and sucrose for dental caries activity testing addresses the complexity and time issues of existing methods, enabling rapid, accurate screening of dental caries susceptibility.
Patent Information
- Application Number
- CN202311186996.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-14
- Publication Date
- 2024-12-20
AI Technical Summary
Existing dental caries detection methods are complex, costly, and time-consuming, making them unsuitable for rapid screening of large populations, especially children and individuals who are difficult to cooperate.
A reagent comprising a redox indicator (resazurin sodium) and a carbon source (sucrose) is used for a dental caries activity test, which includes an inhibitor to prevent false positives, allowing for swab or saliva sampling and culture at 37°C for 15-40 minutes.
The reagent provides quick results within 15-40 minutes, is easy to use, reduces false positives, and is suitable for large-scale screening, accurately distinguishing high-risk from low-risk individuals for dental caries.
Smart Images
Figure CN119162280A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of caries activity test, and specifically to a reagent for caries activity test and a method of use. Background Art
[0002] Dental caries is the most common oral disease that endangers human health. Dental caries can occur as soon as they erupt. The prevalence of dental caries increases with age. The prevalence of dental caries in children aged 5 to 6 is the highest, reaching more than 70% to 80%. Therefore, it is very important to identify the high-risk population for dental caries and implement effective preventive measures.
[0003] Caries activity refers to the body's sensitivity to caries, which can reflect the degree and development speed of caries. Caries activity tests can be divided into three types: methods using saliva as specimens; methods using teeth as specimens; and methods using bacteria as specimens. Currently, the methods using saliva and teeth as specimens are not very sensitive and are rarely used. The method using bacteria as specimens is highly sensitive and reliable, so it is widely used.
[0004] Since the detection methods generally used in China are complex to operate, require high technology and are costly, this study detected cariogenic bacteria and used the biochemical characteristics of cariogenic bacteria to test the caries activity of high-risk populations, with the aim of finding a new, simple and fast method that can effectively screen caries-susceptible populations. This method can achieve a short test time, fast results, and can be directly used in clinical practice without the need for complicated laboratory procedures. It is particularly suitable for the examination of a large number of people, especially for children and other people who are difficult to cooperate.
[0005] Application Contents
[0006] The purpose of the present application is to provide a reagent for a caries activity test and a method of use, which can achieve a short test time and obtain results quickly, can be directly used in clinical practice, does not require complicated steps in the laboratory, and is particularly suitable for the examination of a large number of people, especially for children and other people who are difficult to cooperate.
[0007] To achieve the above-mentioned purpose, the present application provides the following technical solution: a reagent for a caries activity test, comprising a redox indicator 1×10 -7 ~1×10 -3 mol / L and carbon source 1×10 -8 ~0.1mol / L.
[0008] Preferably, the redox indicator comprises resazurin sodium.
[0009] Preferably, the carbon source comprises sucrose.
[0010] Preferably, the dental caries activity test reagent further comprises an inhibitor for inhibiting the growth of non-cariogenic bacteria and reducing false positives of the reagent.
[0011] Preferably, the inhibitor comprises sodium azide. The principle of sodium azide's bacteriostasis is to inhibit the activity of cytochrome oxidase of Gram-negative bacteria, thereby inhibiting the growth of Gram-negative bacteria.
[0012] The present application also provides a method for using a caries activity test reagent, which is used for the above-mentioned caries activity test reagent, comprising the following steps: wiping the teeth with a swab to obtain a bacterial sample, inserting the swab after taking the bacterial sample into the caries activity test reagent for inoculation, removing the swab after elution, and culturing the inoculated caries activity test reagent at 37°C for 15-40 minutes.
[0013] Preferably, wiping teeth with a swab to obtain bacterial samples specifically includes: wiping the buccal side of the left maxillary molar 10 times, the buccal side of the right maxillary molar 10 times, the labial side of the cervical part of the upper front teeth 10 times, and the labial side of the cervical part of the lower front teeth 10 times to obtain bacterial samples.
[0014] Preferably, the elution specifically comprises: eluting the swab inserted into the reagent for the caries activity test by shaking it up and down 10 times and left and right 10 times.
[0015] The present application also provides another method for using a caries activity test reagent, which is used for any of the caries activity test reagents described above, comprising the following steps: collecting a certain amount of saliva sample using a saliva collector, squeezing the saliva sample into the caries activity test reagent for inoculation, and culturing at a constant temperature of 37°C.
[0016] Compared with the prior art, the beneficial effects of this application are:
[0017] (1) Using the dental caries activity test reagent of the present application, the culture effect can be observed after 15-40 minutes of culture. The test time is short and the results are obtained quickly. It can be directly used in clinical practice without the need for complicated laboratory steps. It is particularly suitable for the examination of a large number of people, especially for children and other people who are difficult to cooperate.
[0018] (2) The reagent for the caries activity test of the present application includes an inhibitor, which can inhibit the activity of cytochrome oxidase of Gram-negative bacteria, inhibit the growth of non-cariogenic bacteria, and reduce the false positive of the reagent;
[0019] (3) The caries activity test reagent of the present application is suitable for swab sampling samples and saliva samples as samples for home culture, and the test time of both samples is short and the results are obtained quickly;
[0020] (4) The caries activity test reagent of the present application is easy to use, simple to operate, has a short detection time, significantly shortens the culture time, can quickly provide reference information, has a uniform color after color change, and has more accurate results;
[0021] (5) The caries activity test reagent of the present application is easy to read the culture results of samples with low caries susceptibility, and the results are more accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The color change diagram of high, medium and low risk samples incubated for 5 minutes in Example 4;
[0023] Figure 2 The color change diagram of high, medium and low risk samples in Example 4 after culturing for 10 minutes;
[0024] Figure 3 The color change diagram of high, medium and low risk samples in Example 4 after culturing for 15 minutes;
[0025] Figure 4 This is the color change diagram of high, low and low risk samples cultured for 15 minutes in Example 8;
[0026] Figure 5 This is a color change diagram of four different samples cultured in the formula medium in Comparative Example 1 for 24 hours;
[0027] Figure 6 This is a color change diagram of four different samples cultured in the formula medium in Comparative Example 1 for 30 hours;
[0028] Figure 7 This is a color change diagram of four different samples cultured in the formula medium in Comparative Example 1 for 48 hours;
[0029] Figure 8 This is the color change diagram of the high, medium and low risk samples in comparative example 2 after 15 minutes of incubation;
[0030] Fig. 9 This is the color change diagram of the high, medium and low risk samples in comparative example 2 after culturing for 20 minutes. DETAILED DESCRIPTION
[0031] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.
[0032] Embodiment 1
[0033] A reagent for dental caries activity test, comprising 1×10 -7 ~1×10 -3 mol / L and sucrose 1×10 -8~0.1mol / L, 1×10 -7 ~1×10 -3 mol / L and sucrose 1×10 -8 ~0.1mol / L can be directly mixed to obtain the reagent for the caries activity test.
[0034] Resazurin is a redox indicator. In the metabolism of Gram-positive bacteria, resazurin shows strong sensitivity and changes color to different degrees in an anaerobic environment and in the presence of hydrogen atom donors such as NADH. Under the action of dehydrogenase, hydrogen atoms are transferred, changing resazurin from blue to red to colorless or white. In the study of oral caries activity, Green et al. first used resazurin indicator for bacterial detection in saliva as early as 1960. Later, foreign scholars used resazurin test strips to qualitatively detect bacteria in saliva and clarified the relationship between color change and salivary bacterial levels. The resazurin test results in this article showed that the color change degree of dft and resazurin test, and the level of salivary Streptococcus mutans and the color change degree of resazurin test were positively correlated. With the increase of dft and salivary Streptococcus mutans levels, the color change degree of resazurin test also increased. These results are consistent with the results reported. The color change degree of resazurin test in the high caries group was significantly different from that in the non-caries group. Therefore, the resazurin test has good clinical value and can be promoted and applied as a newer caries activity test method for screening people susceptible to caries.
[0035] Caries activity refers to the body's sensitivity to caries, which can reflect the degree and development speed of caries. Caries activity tests can be divided into three types: methods using saliva as specimens; methods using teeth as specimens; and methods using bacteria as specimens. Currently, the methods using saliva and teeth as specimens are not very sensitive and are rarely used. The methods using bacteria as specimens are highly sensitive and reliable, so they are widely used. The resazurin test determines the caries activity of bacteria in saliva by detecting the redox ability of bacteria in saliva.
[0036] The reagent for the caries activity test is used for monitoring Streptococcus mutans in the oral cavity and screening for caries risk. Sucrose provides the reagent with sufficient carbon source and other nutrients for bacterial growth. Sucrose is consumed during bacterial culture. The reagent contains the indicator sodium resazurin. By detecting the acid-producing ability of salivary cariogenic bacteria and observing the color change of the reagent, the growth of microorganisms can be judged, and the risk of individual caries can be evaluated. The sensitivity of the body to caries can be clarified, and the degree and progression of caries can be accurately reflected. The caries activity of the body can be objectively measured, thereby dividing individuals or groups into those who are highly susceptible to caries and those who are at low risk, formulating corresponding prevention and control measures, determining the intervals for regular inspections and prevention and control, and improving the efficiency of prevention and control work.
[0037] Embodiment 2
[0038] A method for using a dental caries activity test reagent, used for the dental caries activity test reagent of embodiment 1, comprises the following steps:
[0039] A1: Use a swab to swab the buccal side of the left maxillary molar 10 times, the buccal side of the right maxillary molar 10 times, the labial side of the cervical part of the upper anterior teeth 10 times, and the labial side of the cervical part of the lower anterior teeth 10 times to collect bacterial samples;
[0040] A2: Insert the swab after taking the bacteria sample into the caries activity test reagent for inoculation. Shake the swab up and down 10 times and shake it left and right 10 times to wash it out. After washing, take out the swab and discard it;
[0041] A3: Cultivate the inoculated caries activity test reagent at 37°C for 15-40 minutes and observe the culturing effect.
[0042] Embodiment 3
[0043] A method for using a dental caries activity test reagent, used for the dental caries activity test reagent of embodiment 1, comprises the following steps:
[0044] A1: Use a saliva collector to collect 0.8-1.5 ml of saliva for later use;
[0045] A2: squeeze saliva into the reagent for caries activity test for inoculation;
[0046] A3: Mix the reagents for the caries activity test after inoculation at 37°C and let it stand for 15-40 minutes to observe the incubation effect.
[0047] Embodiment 4
[0048] Resazurin sodium 1.06×10 -4 The dental caries activity test reagent composed of 0.1 mol and 0.028 mol of sucrose was used for the test using the method of Example 2. Before collecting the sample, marks 1, 2 and 3 were made on the reagent label. Figure 1-3 As shown, 1 is a high-risk sample, 2 is a medium-risk sample, and 3 is a low-risk sample. Figure 1 The incubation time was 5 min. Figure 2 The incubation time was 10 min. Figure 3 The incubation time was 15 min.
[0049] according to Figure 1-3 It can be seen that for low-risk samples, the reagent color did not change significantly after 5 minutes of incubation. At 10 minutes, the color deepened, and after 15 minutes, it changed from the original blue to purple-pink;
[0050] For medium-risk samples, add the reagent and incubate for 5 minutes. The color of the reagent changes to darker and a light pink precipitates. After 10 minutes, the color changes to purple-pink and turns to pink after 15 minutes.
[0051] For high-risk samples, the reagent was added and incubated for 5 minutes. The color of the reagent changed to purple-pink. After 10 minutes, the color changed to dark pink. After 15 minutes, it turned to pink-white, indicating that the sample was degraded.
[0052] In summary, the color rendering effect of this embodiment is obvious.
[0053] Embodiment 5
[0054] Resazurin sodium 1.06×10 -5 mol and sucrose 2.8×10 -3 The dental caries activity test reagent composed of mol was tested using the method of Example 2. Similarly, the reagent label was marked before the samples were collected, and high, medium and low risk samples were cultured respectively.
[0055] Finally, for low-risk samples, the reagent was added and incubated for 20 minutes. The color of the reagent did not change significantly. At 40 minutes, the color deepened, and after 50 minutes, it changed from the original blue to purple-pink.
[0056] For medium-risk samples, the reagent was added and incubated for 20 minutes. The color of the reagent became darker and a light pink color precipitated. After 40 minutes, the color turned into purple-pink and turned into pink after 50 minutes.
[0057] For high-risk samples, the reagent was added and incubated for 20 minutes. The color of the reagent changed to purple-pink. After 40 minutes, the color changed to dark pink. After 50 minutes, it turned to pink-white, indicating sample degradation.
[0058] In summary, the color development effect of this embodiment is good, but the incubation time is long.
[0059] Embodiment 6
[0060] Resazurin sodium 1.06×10 -6 mol and sucrose 2.8×10 -4 The dental caries activity test reagent composed of mol was tested using the method of Example 2. Similarly, the reagent label was marked before the samples were collected, and high, medium and low risk samples were cultured respectively.
[0061] Finally, we obtained a low-risk sample. After adding the reagent and incubating for 2 hours, the color of the reagent did not change;
[0062] For medium-risk samples, the reagent was added and incubated for 2 hours, and the reagent color did not change;
[0063] For high-risk samples, the reagent was added and incubated for 2 hours, and the color change of the reagent was not obvious.
[0064] In summary, the coloring effect of this embodiment is not obvious.
[0065] Embodiment 7
[0066] Resazurin sodium 1.06×10 -4 The caries activity test reagent was composed of 0.1 mol and 0.019 mol of sucrose. The control group used 1.3 ml of reagent, and only the sucrose concentration was different. Finally, it was found that the photometric value increased, but not significantly. Therefore, the effect of sucrose on the color reaction of the reagent was approximately ignored, and the original sucrose concentration was still used.
[0067] Embodiment 8
[0068] Resazurin sodium 1.06×10 -4 The dental caries activity test reagent composed of 0.1 mol, 0.028 mol sucrose and 0.5 ml / L sodium monoxide was tested using the method of Example 2. Figure 4 As shown, before collecting the samples, mark them on the reagent label, 1, 2 and 3, 1 is a medium-risk sample, 2 is a low-risk sample, 3 is a high-risk sample, and the incubation time is 35 minutes.
[0069] For medium-risk samples in reagent 1, the color changes from blue to pink-purple after adding the reagent and incubating for 35 minutes;
[0070] Reagent 2: For low-risk samples, add the reagent and incubate for 35 minutes, the color changes from blue to light pink-purple;
[0071] Reagent 3: For medium- and high-risk samples, the color changes from blue to pink after adding the reagent and incubating for 35 minutes.
[0072] Comparative Example 1
[0073] The formula of culture medium and improved products based on the Japanese Cariostat method generally includes: trypsin, sucrose, sodium chloride, bromocresol green and bromocresol purple.
[0074] The culture medium inoculated with the above culture medium formula is placed in a 37°C incubator for 24-48 hours. Figure 5-7, All three figures show samples with decreasing risks from left to right, and the three figures respectively show the color changes of the samples for 24-48 hours. Under the same culture time conditions, the high-risk samples tend to be yellow, and the low-risk samples tend to be blue. The tendency to blue indicates less bacterial growth, and the tendency to yellow indicates more bacterial growth. Compared with Example 4 of the present application, Example 4 is cultured for 15 minutes and it is obvious that the high-risk tends to be pink, indicating more bacterial growth, and the low-risk tends to be blue, indicating less bacterial growth. However, it is obvious that the effect of Example 4 is much better than the color development effect of Comparative Example 1, which fully demonstrates that the caries activity test reagent of the present application can significantly shorten the culture time, can quickly evaluate the individual's caries risk, can clarify the body's sensitivity to caries, can accurately reflect the degree of caries and the speed of progression, and objectively measure the body's caries activity, thereby dividing individuals or groups into high-risk susceptible and low-risk caries, formulating corresponding prevention and control measures, determining the interval time for regular inspection and prevention, and improving the efficiency of prevention and control work.
[0075] Comparative Example 2
[0076] The existing resazurin paper method formula is: resazurin sodium, polyvinyl alcohol and sucrose, and the pH is adjusted to 7. After the above culture medium is inoculated, it is placed in a 37°C incubator for cultivation. Figure 8 and 9 As shown, 1, 2, and 3 represent high risk, low risk, and medium risk, respectively. Figure 8 The color chart for 15min culture of high risk, low risk and medium risk is shown in Figure 1. Fig. 9 The color diagram of high-risk, low-risk and medium-risk cultures for 20 minutes. The color of the culture medium changes from blue, purple, pink and light pink according to the bacterial growth. A tendency toward blue indicates less bacterial growth, and a tendency toward pink indicates more bacterial growth. Compared with Example 4, it can be seen that the comparative example three-blade azure paper method is not easy to read. The culture effect of Example 4 is more obvious with the same culture time, which can quickly evaluate the individual's caries risk and improve the efficiency of prevention and treatment.
Claims
1. A reagent for a caries activity test, characterized in that: Including redox indicator 1×10 -7 ~1×10 -3 mol / L and carbon source 1×10 -8 ~0.1mol / L.
2. A dental caries activity test reagent according to claim 1, characterized in that: The redox indicator includes resazurin sodium.
3. A dental caries activity test reagent according to claim 1 or 2, characterized in that: The carbon source includes sucrose.
4. A dental caries activity test reagent according to claim 3, characterized in that: The dental caries activity test reagent also includes an inhibitor for inhibiting the growth of non-cariogenic bacteria and reducing false positives of the reagent.
5. A dental caries activity test reagent according to claim 4, characterized in that: Such inhibitors include sodium azide.
6. A method for using a reagent for a caries activity test, characterized in that: The caries activity test reagent as claimed in any one of claims 1 to 5 comprises the following steps: swabbing the teeth with a swab to obtain a bacterial sample, inserting the swab after the bacterial sample is taken into the caries activity test reagent for inoculation, taking out the swab after elution, and culturing the inoculated caries activity test reagent at 36-38°C.
7. The method for using a dental caries activity test reagent according to claim 6, characterized in that: Swabping teeth to obtain bacterial samples specifically includes: swabbing the buccal side of the left maxillary molar several times, the buccal side of the right maxillary molar several times, the labial side of the cervical part of the upper front teeth several times, and the labial side of the cervical part of the lower front teeth several times to obtain bacterial samples.
8. A method for constructing a reagent for a caries activity test according to claim 6 or 7, characterized in that: The elution specifically includes: eluting the reagent for the caries activity test inserted into the swab by shaking it up and down several times and left and right several times.
9. A method for using a reagent for a caries activity test, characterized in that: The dental caries activity test reagent as claimed in any one of claims 1 to 5 comprises the following steps: collecting a certain amount of saliva sample with a saliva collector, squeezing the saliva sample into the dental caries activity test reagent for inoculation, and culturing at a constant temperature of 36-38°C.