Markers for detecting the risk of dental caries and their uses

Abstract

The present application provides a marker for detecting the risk of dental caries and its use. The present application provides two salivary protein markers, keratin 3 (KRT3) and mucin 21 (MUC21), which are closely associated with the risk of dental caries. The markers of the present application can be used to detect the risk of dental caries non-invasively and inexpensively, thereby achieving accurate prevention and control of dental caries, and ultimately achieving the goal of reducing the incidence of dental caries, especially the incidence of dental caries in young children.

Description

Technical Field

[0001] The present application belongs to the field of detection technology, and specifically, relates to a marker for detecting the risk of dental caries and its use. Background Art

[0002] ECC in young children is a highly prevalent chronic infectious disease with the characteristics of young onset, rapid progression, and wide range of caries. It has adverse effects on the physical and mental health of children, and also imposes a huge economic burden on society. According to the 2010 Global Burden of Disease Study, caries of primary teeth ranks as the tenth largest health problem in the world, affecting more than 621 million children worldwide. An epidemiological survey in my country in 2018 showed that the caries rate in 3-year-old children was as high as 50.8%, which increased to 63.6% at the age of 4 and as high as 71.9% at the age of 5. If the caries risk can be predicted based on simple and non-invasive methods at the age of 3 or even younger, and people at high risk of caries can be screened out, precise prevention and control can be taken. While reducing the caries rate and maintaining children's oral and physical and mental health, it will also greatly reduce the economic burden on families and society.

[0003] Caries is a microbial-mediated disease of dental hard tissues that occurs in the oral saliva microenvironment. The occurrence and development of caries are complexly regulated by the host and microorganisms. As a unique medium involving both host and microbial factors, saliva is an important model for studying the pathogenesis of caries.

[0004] Dental caries is a predictable disease. Previous studies have established several dental caries risk prediction models based on the relative abundance data of oral microorganisms at the genus level. However, due to factors such as horizontal gene transfer and functional redundancy, it is difficult to accurately link species composition with bacterial community function based on studies at the microbial species level. At the same time, the dental caries prediction model based on high-throughput sequencing is complex to construct and difficult to transform into clinical applications that require high operability, convenience, speed, and low cost. Summary of the invention

[0005] In view of the problems existing in the prior art, the present application provides a marker for detecting the risk of dental caries and its use.

[0006] Specifically, this application involves the following aspects:

[0007] 1. Use of keratin 3 (KRT3) and / or mucin 21 (MUC21) in the preparation of a kit for detecting the risk of dental caries in a subject.

[0008] 2. The use according to claim 1, wherein the subject has deciduous dentition, permanent dentition, or mixed dentition.

[0009] 3. The use according to item 1, wherein the risk of dental caries development is the risk of dental caries development within 3 to 12 months from the date of detection.

[0010] 4. The use according to item 1, wherein the subject is a child under 6 years old.

[0011] 5. The use according to item 1, wherein the sample used for detection in the kit is selected from one or more of saliva, tongue swab, buccal mucosa, and dental plaque.

[0012] 6. A kit according to item 5, wherein the saliva is non-stimulated saliva.

[0013] 7. A kit for detecting the risk of dental caries in a subject, comprising reagents for detecting keratin 3 (KRT3) and / or mucin 21 (MUC21).

[0014] 8. A kit according to item 7, wherein the subject has deciduous dentition, permanent dentition, or mixed dentition.

[0015] 9. A kit according to item 7, wherein the subject is a child under 6 years old.

[0016] 10. The kit according to item 7, wherein the sample used for detection in the kit is selected from one or more of saliva, tongue swab, buccal mucosa, and dental plaque.

[0017] 11. A kit according to item 10, wherein the saliva is non-stimulated saliva.

[0018] 12. The kit according to any one of items 7 to 11, wherein the risk of dental caries development is the risk of dental caries development for 3 to 12 months from the date of detection.

[0019] The present application provides two salivary protein markers, keratin 3 (KRT3) and mucin 21 (MUC21) in saliva, which are closely associated with the risk of dental caries. The markers of the present application can be used to non-invasively and cheaply detect the risk of dental caries, thereby achieving accurate prevention and control of dental caries, and ultimately achieving the goal of reducing the incidence of dental caries, especially the incidence of dental caries in young children. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Shows changes in KRT3 levels in saliva samples;

[0021] Figure 2 Shows changes in the amount of MUC21 in saliva samples;

[0022] Figure 3 ROC curve for predicting caries occurrence using salivary KRT3 protein in this prospective cohort;

[0023] Figure 4 ROC curve for predicting dental caries using salivary MUC21 protein in this prospective cohort;

[0024] Figure 5 Shows the changes in KRT3 levels in saliva samples of the new validation cohort;

[0025] Figure 6 Shows changes in MUC21 levels in saliva samples of the new validation cohort;

[0026] Figure 7 ROC curve for predicting caries occurrence using salivary KRT3 protein in the new validation cohort;

[0027] Figure 8 ROC curve for predicting dental caries using salivary MUC21 protein in the new validation cohort. DETAILED DESCRIPTION

[0028] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0029] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0030] "Detection" in the present application refers to any process of observing a marker or marker change (e.g., a change in the content of keratin 3 (KRT3) and / or mucin 21 (MUC21)) in a sample, regardless of whether the marker or marker change is actually detected. In other words, the act of detecting a marker or marker change in a sample is "detection", even if the marker is determined to be absent or below a sensitivity level. Detection can be quantitative, semi-quantitative, or non-quantitative observation, and can be based on comparison with one or more control samples.

[0031] Based on the problems existing in the prior art, the present application provides the use of salivary protein markers (such as keratin 3 (KRT3) and / or mucin 21 (MUC21)) in detecting the risk of dental caries in a subject.

[0032] In a specific embodiment, the present application provides the use of keratin 3 (KRT3) in detecting the risk of dental caries in a subject.

[0033] In a specific embodiment, the present application provides the use of keratin 3 (KRT3) in preparing a kit for detecting the risk of dental caries in a subject.

[0034] In a specific embodiment, the present application provides the use of mucin 21 (MUC21) in detecting the risk of dental caries in a subject.

[0035] In a specific embodiment, the present application provides the use of mucin 21 (MUC21) in preparing a kit for detecting the risk of dental caries in a subject.

[0036] In the above uses, the subjects can be humans of all ages, for example, children, adults, and the elderly.

[0037] In a specific embodiment, the child is under 6 years old.

[0038] In a specific embodiment, the child is 3-4 years old.

[0039] In a specific embodiment, the subject has primary dentition.

[0040] In a specific embodiment, the subject has permanent dentition.

[0041] In a specific embodiment, the subject has mixed dentition.

[0042] In a specific embodiment, the subject is a child with primary dentition.

[0043] In the present application, the risk of dental caries can be the risk of dental caries from 3 to 12 months from the date of detection, for example, it can be the risk of dental caries from 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months from the date of detection, or any time between these times.

[0044] In a specific embodiment, the risk of dental caries development is the risk of dental caries development within 6 to 12 months from the date of detection.

[0045] The sample used for detection in the kit can be selected from one or more of saliva, tongue swab, buccal mucosa, and dental plaque.

[0046] The present application also provides a kit for detecting the risk of dental caries in a subject, comprising reagents for detecting keratin 3 (KRT3) and / or mucin 21 (MUC21).

[0047] In a specific embodiment, the kit includes a reagent for detecting keratin 3 (KRT3).

[0048] In a specific embodiment, the kit includes a reagent for detecting mucin 21 (MUC21).

[0049] In a specific embodiment, the kit includes reagents for detecting keratin 3 (KRT3) and mucin 21 (MUC21).

[0050] The kit also includes a buffer required for detecting salivary protein markers.

[0051] Furthermore, the kit also includes a container for containing patient samples. And, the kit also includes instructions for use and interpretation of test results.

[0052] In the above kit, the subjects can be humans of all ages, for example, children, adults, and the elderly.

[0053] In a specific embodiment, the child is under 6 years old.

[0054] In a specific embodiment, the child is 3-4 years old.

[0055] In a specific embodiment, the subject has primary dentition.

[0056] In a specific embodiment, the subject has permanent dentition.

[0057] In a specific embodiment, the subject has mixed dentition.

[0058] In a specific embodiment, the subject is a child with primary dentition.

[0059] In the present application, the risk of dental caries can be the risk of dental caries from 3 to 12 months from the date of detection, for example, it can be the risk of dental caries from 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months from the date of detection, or any time between these times.

[0060] In a specific embodiment, the risk of dental caries development is the risk of dental caries development within 6 to 12 months from the date of detection.

[0061] The sample used for detection in the kit can be selected from one or more of saliva, tongue swab, buccal mucosa, and dental plaque.

[0062] The saliva includes non-irritated saliva and irritated saliva. The sample of the present application is preferably non-irritated saliva, such as non-irritated saliva in a resting state.

[0063] The present application also provides a method for detecting the risk of dental caries in a subject, the method comprising the following steps:

[0064] Extracting samples to be tested;

[0065] contacting the sample to be tested with a reagent for detecting keratin 3 (KRT3) and / or mucin 21 (MUC21) to determine the content of keratin 3 (KRT3) and / or mucin 21 (MUC21);

[0066] The risk of caries was determined based on the comparison of keratin 3 (KRT3) and / or mucin 21 (MUC21) levels with specified thresholds.

[0067] In a specific embodiment, the designated threshold value of keratin 3 (KRT3) is 407.075 μg / L, and a value above this threshold value indicates a risk of developing dental caries.

[0068] In a specific embodiment, the designated threshold value of mucin 21 (MUC21) is 46.822 μg / L, and a value above this threshold value indicates a risk of developing dental caries.

[0069] In the above method, the subject can be humans of all ages, for example, children, adults, and the elderly.

[0070] In a specific embodiment, the child is under 6 years old.

[0071] In a specific embodiment, the child is 3-4 years old.

[0072] In a specific embodiment, the subject has primary dentition.

[0073] In a specific embodiment, the subject has permanent dentition.

[0074] In a specific embodiment, the subject has mixed dentition.

[0075] In a specific embodiment, the subject is a child with primary dentition.

[0076] In the present application, the risk of dental caries can be the risk of dental caries from 3 to 12 months from the date of detection, for example, it can be the risk of dental caries from 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months from the date of detection, or any time between these times.

[0077] In a specific embodiment, the risk of dental caries development is the risk of dental caries development within 6 to 12 months from the date of detection.

[0078] The sample to be tested can be selected from one or more of saliva, tongue swab, buccal mucosa, and dental plaque.

[0079] In a specific embodiment, the sample to be tested is saliva, preferably non-stimulated saliva.

[0080] The applicant of the present application found that the two markers, keratin 3 (KRT3) and mucin 21 (MUC21), are closely associated with the risk of dental caries. The content of keratin 3 (KRT3) or mucin 21 (MUC21) in subjects with dental caries in the future is significantly different from that in subjects without dental caries. The markers of the present application can be used to detect the risk of dental caries non-invasively and cheaply, so that accurate prevention and control of dental caries can be achieved, and ultimately the goal of reducing the incidence of dental caries, especially the incidence of dental caries in young children, can be achieved.

[0081] Example

[0082] Example 1

[0083] 1. Non-stimulated saliva sample collection and quantitative proteomic testing for a prospective cohort study

[0084] Based on a prospective cohort study, 38 healthy, caries-free children aged 3-4 years were followed up for 12 months and divided into a caries-acquired group (CA) (21 cases) and a caries-free group (CF) (17 cases) according to whether caries occurred as an outcome. Quantitative proteomic analysis of resting, non-stimulated saliva samples was performed using data-independent (DIA) mass spectrometry acquisition at baseline and at 12 months of follow-up.

[0085] 2. Analysis of proteomic test results and screening of protein biomarkers that can predict the occurrence of dental caries

[0086] According to the outcome of caries or not after 12 months, the baseline individuals were grouped and differential proteome analysis was performed, aiming to find protein biomarkers that can predict caries 1 year before the onset of caries. In order to facilitate the development of subsequent detection kits, in the differential protein analysis of the two groups at baseline, the focus was on the proteins that were upregulated in the caries group compared with the healthy group, a total of 93. After that, the inter-group trend analysis of the differential proteins was further performed, focusing on the proteins whose relative amounts were still upregulated after 12 months of caries; at the same time, the screening was performed according to the degree of data integrity: the original identification rate of the protein in all samples was stipulated to be higher than 80%. Finally, according to the above criteria, two protein markers were screened, namely keratin 3 (KRT3) and mucin 21 (MUC21).

[0087] 3. Validation of the predictive accuracy of salivary protein markers using enzyme-linked immunosorbent assay (ELISA) in the trial cohort samples

[0088] Specific steps: The saliva samples of the two groups of children in the cohort at the baseline (0 months) were centrifuged at 10,000 rpm at 4°C, and the supernatant was used for ELISA experiments, and diluted and added according to the operating instructions of the human keratin 3 (KRT3) and human mucin 21 (MUC21) ELISA kits (Jiangsu ELISA). First, the saliva samples were incubated at 37°C for 30 minutes, and the concentrated solution was thoroughly washed after dilution, treated with enzymes, warmed, washed and colored, and the stop solution was added to stop the reaction. The assay was performed within 15 minutes after the addition of the stop solution, and the absorbance was measured at a wavelength of 450 nm using an enzyme marker (Biotech, America), and a standard curve was derived to read the content of KRT3 and MUC21 in each sample.

[0089] The detection results of KRT3 are as follows Figure 1 As shown, the KRT3 protein level of the caries-free group (CF) at baseline was 326.18±19.78μg / L, the lowest value was 305.90μg / L, and the highest value was 367.32μg / L; the KRT3 protein level of the caries group (CA) at baseline was 429.96±17.14μg / L, the lowest value was 406.89μg / L, and the highest value was 461.34μg / L. The KRT3 protein level in the saliva of children with caries was significantly higher than that of healthy children 12 months before they became ill.

[0090] The detection results of MUC21 are as follows Figure 2 As shown in the figure, the MUC21 protein level of the caries-free group (CF) at baseline was 43.88±5.34μg / L, the lowest value was 37.90μg / L, and the highest value was 52.90μg / L; the MUC21 protein level of the caries group (CA) at baseline was 51.78±2.26μg / L, the lowest value was 46.88μg / L, and the highest value was 54.97μg / L. In the first 12 months of their illness, the MUC21 protein level in the saliva of children with caries was significantly higher than that of healthy children.

[0091] The ROC curve was drawn based on the test results of the two proteins and the actual dental caries situation of the prospective cohort (17 of the 38 children remained healthy and caries-free after 12 months, and 21 developed dental caries). Figure 3 As shown, the ROC curve of MUC21 is as follows Figure 4 The results showed that the prediction accuracy of KRT3 protein was 100%, and the prediction accuracy of MUC21 protein was 87.1%.

[0092] Example 2 Validation of salivary protein markers in a wide range of populations

[0093] 1. Collection of Unstimulated Saliva Samples from the Validation Cohort

[0094] A total of 75 healthy, caries-free children aged 3-4 years were followed up for 6 months. 2 mL of unstimulated saliva samples were collected at baseline. The children were divided into a caries-acquired group (CA) of 25 cases and a caries-free group (CF) of 50 cases according to whether caries occurred as an outcome.

[0095] 2. Enzyme-linked immunosorbent assay (ELISA) was used to verify the predictive accuracy of salivary protein markers in the validation cohort samples.

[0096] Specific steps: The saliva samples of the two groups of children in the cohort at the baseline (0 months) were centrifuged at 10,000 rpm at 4°C, and the supernatant was used for ELISA experiments, and diluted and added according to the operating instructions of the human keratin 3 (KRT3) and human mucin 21 (MUC21) ELISA kits (Jiangsu ELISA). First, the saliva samples were incubated at 37°C for 30 minutes, and the concentrated solution was thoroughly washed after dilution, treated with enzymes, warmed, washed and colored, and the stop solution was added to stop the reaction. The assay was performed within 15 minutes after the addition of the stop solution, and the absorbance was measured at a wavelength of 450 nm using an enzyme marker (Biotech, America), and a standard curve was derived to read the content of KRT3 and MUC21 in each sample.

[0097] The detection results of KRT3 are as follows Figure 5 As shown, the KRT3 protein level of the caries-free group (CF) at baseline was 381.37±16.90μg / L, the lowest value was 354.98μg / L, and the highest value was 409.68μg / L; the KRT3 protein level of the caries group (CA) at baseline was 408.57±16.56μg / L, the lowest value was 381.68μg / L, and the highest value was 439.35μg / L. In the first 6 months of caries, the KRT3 protein level in the saliva of children with caries was significantly higher than that of healthy children.

[0098] The detection results of MUC21 are as follows Figure 6As shown in the figure, the MUC21 protein level of the caries-free group (CF) at baseline was 45.61±2.35μg / L, the lowest value was 41.58μg / L, and the highest value was 51.04μg / L; the MUC21 protein level of the caries group (CA) at baseline was 47.57±2.34μg / L, the lowest value was 43.02μg / L, and the highest value was 51.79μg / L. In the first 6 months of caries, the MUC21 protein level in the saliva of children with caries was significantly higher than that of healthy children.

[0099] The ROC curve was drawn based on the test results of the two proteins and the actual caries situation of the validation cohort (6 months later, 50 of the 75 children remained healthy and caries-free, and 25 developed caries). Figure 7 As shown, the ROC curve of MUC21 is as follows Figure 8 The results showed that the prediction accuracy of KRT3 protein was 86.4%, and the prediction accuracy of MUC21 protein was 71.3%.

Claims

1. Use of a reagent for detecting KRT3 and / or MUC21 in the preparation of a kit for detecting the risk of dental caries in a subject, wherein the risk of dental caries is the risk of dental caries 3 to 12 months from the date of detection, the subject is a child under 6 years old, and the sample used for detection in the kit is saliva.

2. The use according to claim 1, wherein the subject has primary dentition or mixed dentition.

3. The use according to claim 1, wherein the saliva is non-irritating saliva.

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