Application of streptococcus mutans NRPS / PKS gene cluster subtype, primer pair, kit and system

Abstract

The invention relates to the field of dental caries, and discloses application of a streptococcus mutans NRPS / PKS gene cluster subtype, a primer pair, a kit and a system. Different NRPS / PKS gene subtypes exist in streptococcus mutans, the molecular copy number of the different NRPS / PKS gene subtypes is highly related to occurrence of caries, the different NRPS / PKS gene subtypes can be used as a caries occurrence risk prediction index, and a new direction is provided for prognosis judgment after prevention and treatment of caries.

Description

Technical Field

[0001] The present invention relates to the field of dental caries, and specifically relates to the application of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of products for evaluating the risk of dental caries occurrence, the application of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of products for evaluating and determining whether the risk of dental caries occurrence through clinical risk assessment should be reclassified, a primer pair, a kit, and a system for judging the occurrence risk. Background Art

[0002] Although the techniques for the examination, treatment, and prevention of dental caries have been improved, it is still prevalent in people of all age groups. If not treated correctly and in a timely manner, dental caries may cause permanent damage.

[0003] Traditional methods for examining dental caries include using dental exploration equipment, X-ray imaging for auxiliary examination, visual and tactile probing methods, etc. For the examination methods using the above, the results are often affected by many factors such as the experience of the operator, the severity of the disease condition, the observation situation, etc., and there may also be a risk of damaging the teeth. By the time dental caries is obvious under visual and tactile examinations, the disease is usually in the attack stage and requires filling. If not treated in a timely manner, it may cause lesions such as pulpitis and apical periodontitis.

[0004] Currently, there is still a lack of effective methods for predicting the risk of dental caries occurrence, and it is still impossible to prevent the occurrence of dental caries in a timely and accurate manner, or to conduct a health assessment of the treated teeth. Summary of the Invention

[0005] The purpose of the present invention is to overcome the problem that the prior art cannot effectively predict the risk of dental caries occurrence, and to provide the application of Streptococcus mutans NRPS / PKS gene cluster subtypes, as well as a primer pair, a kit, and a system. The technical solution provided by the present invention has the advantage of quantitatively judging the risk of dental caries occurrence, and provides a new and simple operation detection tool for predicting the risk of dental caries occurrence.

[0006] To achieve the above purpose, in the first aspect of the present invention, there is provided the application of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of products for evaluating the risk of dental caries occurrence.

[0007] In the second aspect of the present invention, there is provided the application of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of products for evaluating and determining whether the risk of dental caries occurrence through clinical risk assessment should be reclassified.

[0008] In the third aspect of the present invention, there is provided a primer pair for conventional PCR and / or real-time fluorescence quantitative PCR, and the primer pair has the ability to specifically and quantitatively amplify Streptococcus mutans NRPS / PKS gene cluster subtypes.

[0009] In the fourth aspect of the present invention, there is provided a kit for evaluating the risk of dental caries by detecting the subtypes of the Streptococcus mutans NRPS / PKS gene cluster, and the kit includes the primer pair described in the third aspect.

[0010] In the fifth aspect of the present invention, there is provided a system for judging the risk of dental caries, and the system includes:

[0011] A receiving unit, configured to receive an instruction for evaluating the risk of dental caries;

[0012] A detecting unit, configured to detect the signal output of the amplicons obtained by amplifying a sample based on the instruction;

[0013] Wherein, the detecting unit includes a sample amplification area, and the primer pair described in the third aspect or the kit described in the fourth aspect is arranged in the sample amplification area.

[0014] By the above technical solutions, the present invention can at least achieve the following beneficial effects:

[0015] Currently, there is a lack of effective methods for predicting the risk of dental caries. The present invention discovers that the molecular copy numbers of different NRPS / PKS gene subtypes in Streptococcus mutans are highly correlated with the occurrence of dental caries, and can be used as an indicator for predicting the risk of dental caries, providing a new method for the prevention of dental caries and the prognosis determination after treatment.

[0016] The present invention first proposes a method for predicting the risk of dental caries based on the subtypes of the Streptococcus mutans NRPS / PKS gene cluster. The products provided by the present invention make the operation of predicting the risk of dental caries simpler, and have the advantages of being able to judge the risk of dental caries through both qualitative and quantitative methods, providing a new type of detection tool for predicting the risk of dental caries. Description of the Drawings

[0017] Figure 1 is the decayed, missing, and filled teeth index (dmft) of the primary teeth in the SECC group in Example 1.

[0018] Figure 2 is a comparison chart of the detection numbers of each subtype of the NRPS / PKS gene cluster in the caries-free group and the SECC group in Example 1.

[0019] Figure 3 is a quantitative relationship chart between the molecular copy numbers of each subtype of the NRPS / PKS gene cluster detected by real-time fluorescence quantitative PCR (qPCR) and the number of amplification cycles in Example 1.

[0020] Figure 4 is a comparison chart of the agarose gel electrophoresis results of the conventional PCR and qPCR products of each subtype of the NRPS / PKS gene cluster in Example 1.

[0021] Figure 5 It is a ROC curve analysis result diagram of each subtype of the NRPS / PKS gene cluster in Example 1. Figure 6 It is a result diagram of the ROC combined analysis for predicting the risk of SECC occurrence of each subtype of the NRPS / PKS gene cluster in Example 1. Detailed implementation manners

[0022] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0023] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

[0024] Through extensive research, the inventors of the present invention found that some subtypes of the Streptococcus mutans NRPS / PKS gene cluster are associated with the occurrence of dental caries. By detecting specific subtypes of the Streptococcus mutans NRPS / PKS gene cluster in the oral flora, the risk of dental caries occurrence in a subject can be evaluated. Quantitative amplification of these Streptococcus mutans NRPS / PKS gene cluster subtypes can more simply and intuitively judge the risk of dental caries occurrence in a subject through the qPCR results. On this basis, combined with other detection means (such as existing traditional detection means in the art, etc.), the risk of dental caries occurrence in a subject can be judged more scientifically and systematically, and guidance for the subject's future oral care and related treatments can be provided.

[0025] Based on this, a first aspect of the present invention provides the use of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of a product for evaluating the risk of dental caries occurrence.

[0026] Preferably, the NRPS / PKS gene cluster subtype comprises at least one of Smul1335, UA140, and Orf-H.

[0027] Preferably, the dental caries is selected from severe early childhood caries (SECC).

[0028] The inventors of the present invention found that when using the primers of the above nucleotide sequences respectively for real-time fluorescence quantitative PCR detection, the expressions of Smul1335, UA140, and Orf-H are significantly statistically significant (P < 0.001) in the caries and caries-free groups.

[0029] In the present invention, the product may be of the types existing in the art, preferably primers, systems or kits.

[0030] The second aspect of the present invention provides the use of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of a product for evaluating and determining whether the risk of dental caries occurrence determined by clinical risk assessment should be reclassified.

[0031] The preferred Streptococcus mutans NRPS / PKS gene cluster subtypes and products are as described in the first aspect, and will not be elaborated herein.

[0032] Preferably, the dental caries is selected from severe early childhood caries (SECC).

[0033] The third aspect of the present invention provides a primer pair for conventional PCR and / or real-time fluorescence quantitative PCR, and the primer pair has the ability to specifically quantitatively amplify Streptococcus mutans NRPS / PKS gene cluster subtypes.

[0034] The preferred Streptococcus mutans NRPS / PKS gene cluster subtypes are as described in the first aspect, and will not be elaborated herein.

[0035] Preferably, the primer pair for Smu1335 includes a forward primer having the nucleotide sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 7 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 2 and / or SEQ ID NO: 8.

[0036] Preferably, the primer pair for UA140 includes a forward primer having the nucleotide sequence shown in SEQ ID NO: 3 and / or SEQ ID NO: 9 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 4 and / or SEQ ID NO: 10.

[0037] Preferably, the primer pair for Orf-H includes a forward primer having the nucleotide sequence shown in SEQ ID NO: 5 and / or SEQ ID NO: 11 and a reverse primer having the nucleotide sequence shown in SEQ ID NO: 6 and / or SEQ ID NO: 12.

[0038] According to the present invention, the synthesis method of the primer pair with known sequences is well known to those skilled in the art. For example, it can be synthesized by entrusting a biotechnology company (such as Sangon Biotech Co., Ltd.), so it will not be elaborated herein.

[0039] The above primer pair can be used to evaluate the risk of dental caries occurrence, or to evaluate and determine whether the risk of dental caries occurrence determined by clinical risk assessment should be reclassified.

[0040] The fourth aspect of the present invention provides a kit for evaluating the risk of dental caries by detecting the subtype of the Streptococcus mutans NRPS / PKS gene cluster, and the kit includes the primer pair described in the third aspect.

[0041] Preferably, the kit may further include PCR amplification reagents.

[0042] In the present invention, as long as the primer pair is the above-mentioned primer pair, the object of the present invention can be achieved. The PCR amplification reagent refers to the conventional reagents used for PCR amplification of primers and templates, and there is no special limitation on the PCR amplification reagent. For example, the PCR amplification reagent may include a PCR buffer, a DNA polymerase, and dNTPs. In the PCR amplification reagent, the types and concentrations of the above substances are the types and concentrations commonly used in the art and can all be obtained through commercial purchase.

[0043] Specifically, the PCR amplification reagent can also be set according to the actual detection requirements. For example, when performing quantitative detection, the PCR amplification reagent can be a DNA polymerase, a PCR reaction buffer, MgCl2, dNTPs, and a fluorescent quantitative dye, such as SYBR Green. The concentrations of these components can be the concentrations commonly used in the art, and they can exist in the kit alone or in the form of a Premix. Among them, Premix refers to a composite reagent prepared by premixing all or part of the PCR amplification reagents. Compared with separately storing and using the above various PCR amplification reagents, providing the PCR amplification reagent in the form of a Premix can simplify the operation. The present invention has no particular limitation on the source of the PCR amplification reagent, especially the amplification reagent provided in the form of a Premix. It can either be configured according to the actual situation or directly obtained through commercial purchase or customization. For example, it can be a Premix ( qPCR Kit) purchased from PROMEGA with the product number A6001.

[0044] In the present invention, considering that DNA extraction operation is required before PCR amplification, preferably, the kit further includes DNA extraction reagents. The DNA extraction reagents can be various reagents commonly used in the art for DNA extraction, such as Tris-saturated phenol, phenol / chloroform / isoamyl alcohol (25:24:1), RNase, isopropanol, and sodium acetate. In addition, the concentrations of the above various DNA extraction reagents can also be the concentrations commonly used in the art.

[0045] In the present invention, when performing qualitative detection (such as conventional PCR detection), electrophoresis can be used for qualitative detection. The electrophoresis can be agarose gel electrophoresis. The method of performing agarose gel electrophoresis on the PCR product is well known to those skilled in the art and will not be described in detail here.

[0046] Among them, the control sample preferably includes a positive control sample (for example, the present invention provides gene fragments of each subtype of the NRPS / PKS gene cluster of Streptococcus mutans as a positive control) and a negative control sample (such as sterile MilliQ water). If the target band appears in the positive control and the target band does not appear in the negative control, the amplification is considered to be effective. If the positive control does not appear a band or the negative control appears a target band, the amplification is considered to be invalid and needs to be re-amplified.

[0047] In the present invention, when performing real-time fluorescence quantitative PCR detection, preferably, the control sample preferably includes a positive control sample (for example, a target gene fragment of a standard with a certain number of molecules provided by the present invention as a positive control) and a negative control sample (such as sterile MilliQ water). If within 20-50 cycles, the positive control can produce a fluorescent signal, while the negative control does not produce a fluorescent signal, then the amplification is considered to be effective. If the positive control does not produce a fluorescent signal or the negative control produces a fluorescent signal, then the amplification is considered to be invalid and needs to be re-amplified.

[0048] The standard can be a gene fragment of each subcluster of the corresponding Streptococcus mutans NRPS / PKS with a certain number of molecules, which can be diluted to 10 with sterile MilliQ water. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Concentration gradient of copies / μl.

[0049] Existing PCR detection methods can be used in the present invention. Many available biological literatures also have extended discussions on PCR and related amplification methods, and those skilled in the art can operate according to the relevant literatures.

[0050] The conditions for the real-time fluorescence PCR amplification can be conventional PCR amplification conditions. Preferably, the PCR amplification system is pre-denatured at 94 - 96 °C for 1 - 3 min first; then 35 - 45 cycles are carried out under the following program: 93 - 95 °C, 25 - 35 s, 55 - 65 °C, 45 - 75 s. More preferably, the PCR amplification system is pre-denatured at 94.5 - 95.5 °C for 1.5 - 2.5 min first; then 35 - 45 cycles are carried out under the following program: 93.5 - 94.5 °C, 28 - 32 s, 58 - 62 °C, 55 - 65 s.

[0051] The kit can be used to evaluate the risk of dental caries occurrence, or to evaluate and determine whether the risk of dental caries occurrence through clinical risk assessment should be reclassified. Preferably, the dental caries is selected from severe early childhood caries (SECC).

[0052] The fifth aspect of the present invention provides a system for judging the risk of dental caries occurrence, which system comprises:

[0053] A receiving unit, configured to receive an instruction for evaluating the risk of dental caries occurrence;

[0054] A detecting unit, based on the instruction, detecting the signal output of the amplicons obtained by amplifying a sample;

[0055] Wherein, the detecting unit includes a sample amplification area, and in the sample amplification area, the primer pair as described above or the kit as described above is provided.

[0056] The preferred Streptococcus mutans NRPS / PKS gene cluster subtypes are as described in the first aspect, and the preferred primer pairs are as described in the third aspect, which will not be elaborated here.

[0057] In the present invention, the sample includes but is not limited to samples derived from a subject.

[0058] Preferably, the sample includes genomic DNA, cDNA, RNA, amplified genomic DNA, amplified cDNA or amplified RNA.

[0059] The system provided by the present invention can analyze the detection result and judge the risk of dental caries occurrence of the subject according to the analysis result. In the present invention, either a single subtype among them can be analyzed, or a combined analysis of two or three subtypes can be carried out.

[0060] In order to obtain a more accurate judgment result, according to the preferred embodiment of the present invention, wherein, the system further includes an analysis unit, and the analysis unit jointly judges the risk of dental caries occurrence by analyzing the gene copy numbers of Smu1335, UA140 and Orf-H subtypes obtained by the detecting unit.

[0061] The various units of the system can be integrated or exist independently.

[0062] The system can be a computer-executed system.

[0063] Preferably, the dental caries disease is selected from severe early childhood caries (SECC).

[0064] Using the primer pairs, kits and systems involved in the above content of the present invention, it can be used to evaluate the risk of dental caries disease occurrence, and provide a basis for the prevention of dental caries and the prognosis determination after treatment.

[0065] In the present invention, Streptococcus mutans can be classified using NRPS / PKS gene cluster subtypes (Smu1335, UA140, and Orf-H). Primers designed for NRPS / PKS gene cluster subtypes and polymerase chain reaction (PCR) standards for NRPS / PKS gene cluster subtypes are used to predict and determine the risk of dental caries disease occurrence based on real-time quantitative fluorescence PCR detection. Analyzing and identifying dental plaque according to this method can obtain the probability of dental caries disease occurrence risk, providing a basis for the prevention of dental caries and the prognosis determination after treatment.

[0066] In the present invention, the method for evaluating the risk of dental caries disease occurrence preferably includes:

[0067] (1) DNA extraction: Extract the genomic DNA of oral flora;

[0068] (2) PCR amplification: Using the genomic DNA obtained in step (1) as a template, perform real-time fluorescence quantitative PCR amplification on the sequence of Streptococcus mutans NRPS / PKS gene cluster subtypes using primer pairs specific for amplifying Streptococcus mutans NRPS / PKS gene cluster subtypes;

[0069] (3) Detection: Perform real-time fluorescence quantitative PCR detection on the products of real-time fluorescence quantitative PCR amplification, and judge the susceptibility of dental caries disease occurrence according to the detection results.

[0070] In a preferred embodiment of the present invention, real-time quantitative fluorescence PCR detection is used to evaluate the risk of dental caries disease occurrence. The method includes: extracting the genomic DNA of oral flora; using the obtained genomic DNA as a template, and performing real-time fluorescence quantitative PCR amplification on the sequence of Streptococcus mutans NRPS / PKS gene cluster subtypes using primer pairs.

[0071] Preferably, the method further includes performing statistical analysis on NRPS / PKS gene cluster subtypes (Smu1335, UA140, and Orf-H), plotting a multi-index combined ROC curve for each gene subtype, and determining at least one of the optimal screening positive cut-off value, sensitivity, and specificity by analyzing the area under the curve. Preferably, the method further includes evaluating the risk of dental caries occurrence in the subject based on the results of the above analysis.

[0072] Preferably, the primer pair is selected from at least one pair of SEQ ID No:1 and SEQ ID No:2, SEQ ID No:3 and SEQ ID No:4, and SEQ ID No:5 and SEQ ID No:6.

[0073] Preferably, the primer pair is selected from at least one pair of SEQ ID No:7 and SEQ ID No:8, SEQ ID No:9 and SEQ ID No:10, and SEQ ID No:11 and SEQ ID No:12.

[0074] In the present invention, a specific standard product can be used as a quantitative reference, and the susceptibility to dental caries occurrence can be judged based on the number of molecules of the NRPS / PKS gene cluster subtype.

[0075] Preferably, the judgment criteria include: when the test result of the sample to be tested is greater than or equal to the optimal screening positive cut-off value (cut-off value), it is judged as positive; when it is less than the optimal screening positive cut-off value, it is judged as negative. The cut-off value is judged based on the optimal cut-off point (Associated criterion) generated by Medcalc software. If the test result is greater than this optimal cut-off point, that is, greater than the cut-off value, it can be considered that the subject is a dental caries susceptible person (that is, it is considered that the subject has a risk of dental caries occurrence).

[0076] Preferably, the oral flora is obtained from dental plaque on the tooth surface.

[0077] The system can be used to evaluate the risk of dental caries occurrence, or to evaluate and determine whether the risk of dental caries occurrence determined by clinical risk assessment should be reclassified.

[0078] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention exemplarily, and are not used to limit the present invention.

[0079] In the following examples, unless otherwise specified, the reagents and equipment involved can be obtained through commercial purchase.

[0080] Example 1

[0081] This example is used to illustrate the conventional PCR and real-time fluorescence quantitative PCR detection methods

[0082] 1. Analyze the case samples of the clinically carious group (severe early childhood caries (SECC) group, 143 cases) and the caries-free group (197 cases) using conventional PCR detection. The statistical details of the patients in the SECC group and the caries-free group are shown in Table 1. Figure 1 The decayed, missing, and filled teeth index (dmft) of the primary teeth in the SECC group is shown.

[0083] Table 1

[0084]

[0085] The specific method is as follows:

[0086] A. DNA extraction: Extract the genomic DNA of the oral flora;

[0087] B. PCR reaction: Using the sequences of the NRPS / PKS gene cluster subtypes (Smu1335, UA140, and Orf-H) as the detection indicators, and the oral flora DNA as the template, use the primers shown in Table 2 to perform conventional PCR and qPCR amplifications respectively.

[0088] Table 2 PCR primers for NRPS / PKS gene cluster subtypes (Smu1335, UA140, and Orf-H)

[0089]

[0090]

[0091] Preliminarily analyze the conventional PCR amplification products by agarose gel electrophoresis. The results are shown in Figure 2 . It can be seen from the figure that by counting the number of positive cases with specific bands amplified, it is found that there are statistically significant differences in the NRPS / PKS gene cluster subtypes Smu1335, UA140, and Orf-H between the caries-free and SECC groups (P < 0.05).

[0092] 2. Analyze the case samples of the clinically carious group (72 cases) and the caries-free group (72 cases) using real-time fluorescence quantitative PCR (qPCR) detection.

[0093] The real-time fluorescence quantitative PCR reaction system is as follows: 2×SYBR GREEN Premix 10 μL, 0.5 μL each of the upstream and downstream primers (set three replicate real-time quantitative fluorescence PCR reactions for each primer pair, and the specific primer sequences are shown in Table 2), 1 μL of DNA template, and add dH2O to 20 μL; The PCR reaction program is: pre-denaturation at 95°C for 2 min; 94°C, 30 s; 60°C, 1 min, for 40 cycles.

[0094] By performing qPCR under the above conditions with different initial nucleic acid molecule numbers and cycle numbers, qPCR standard curves for each gene subtype were plotted. For details, see Figure 3 . It can be seen from the figure that as the initial molecule number increases, the cycle number decreases accordingly.

[0095] Figure 4 The agarose gel electrophoresis detection results of PCR and qPCR products of a sample in the caries group are shown. It can be seen from the figure that amplification can be achieved by PCR or qPCR, and the products Smu1335, UA140, and Orf-H obtained by two different PCR methods are all of different sizes.

[0096] Fusion genes containing specific copy numbers of each NRPS / PKS gene cluster subtype were used as standards for real-time quantitative fluorescence PCR detection. Through the qPCR detection results, ROC analysis of the copy numbers of Smu1335, UA140, and Orf-H and the risk of SECC occurrence was performed.

[0097] Furthermore, ROC curve analysis of Smu1335, UA140, and Orf-H was performed using MedCalc software respectively, generating a logical prediction probability value curve (LOGREGR_Pred1), and calculating the area under the probability value curve. Then, the optimal cut-off value was determined accordingly. Values greater than this optimal screening positive cut-off value were judged as being prone to SECC. For details, see Figure 5 .

[0098] Joint ROC analysis of Streptococcus mutans NRPS / PKS gene cluster subtypes Smu1335, UA140, and Orf-H and the risk of SECC occurrence was performed. Logistic regression analysis showed that Smu1335, UA140, and Orf-H were related to the occurrence of SECC, and the p-values in the caries-free group and the SECC group were 0.0011, 0.0346, and 0.0198 respectively. Furthermore, MedCalc software was used to perform joint ROC curve analysis of multiple indicators of Smu1335, UA140, and Orf-H, generating a logical prediction probability value curve (LOGREGR_Pred1), and calculating the area under the probability value curve to be 0.942. Then, according to Medcalc software, the optimal cut-off value (Associated criterion) was determined to be 0.45. At this time, the sensitivity was 91.89% and the specificity was 88.24%. Values greater than this optimal screening cut-off value could be judged as being prone to SECC. For details, see Figure 6 .

[0099] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. Use of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of a product for assessing the risk of dental caries occurrence.

2. The application according to claim 1, wherein The NRPS / PKS gene cluster subtype comprises at least one of Smul1335, UA140, and Orf-H.

3. Use of Streptococcus mutans NRPS / PKS gene cluster subtypes in the preparation of a product for assessing and determining whether the risk of dental caries occurrence determined by clinical risk assessment should be reclassified.

4. The application according to claim 3, wherein, The NRPS / PKS gene cluster subtype comprises at least one of Smul1335, UA140, and Orf-H.

5. A primer pair for conventional PCR and / or real-time fluorescence quantitative PCR, characterized in that, The primer pair has the ability to specifically quantitatively amplify the Streptococcus mutans NRPS / PKS gene cluster subtype.

6. The primer pair for conventional PCR and / or real-time fluorescence quantitative PCR according to claim 5, wherein, The NRPS / PKS gene cluster subtype comprises at least one of Smu1335, UA140, and Orf-H.

7. The primer pair for conventional PCR and / or real-time fluorescence quantitative PCR according to claim 5 or 6, wherein, The primer pair for Smu1335 includes a forward primer having the nucleotide sequence shown in SEQ ID NO:1 and / or SEQ ID NO:7 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:2 and / or SEQ ID NO:8; and / or The primer pair for UA140 includes a forward primer having the nucleotide sequence shown in SEQ ID NO:3 and / or SEQ ID NO:9 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:4 and / or SEQ ID NO:10; and / or The primer pair for Orf-H includes a forward primer having the nucleotide sequence shown in SEQ ID NO:5 and / or SEQ ID NO:11 and a reverse primer having the nucleotide sequence shown in SEQ ID NO:6 and / or SEQ ID NO:

12.

8. A kit for assessing the risk of dental caries by detecting the subtypes of the Streptococcus mutans NRPS / PKS gene cluster, characterized in that, The kit includes the primer pair according to any one of claims 5-8.

9. A system for determining the risk of dental caries, characterized in that, The system includes: A receiving unit for receiving an instruction to assess the risk of dental caries occurrence; A detection unit for detecting, based on the instruction, the signal output of the amplicon obtained by amplifying a sample; Wherein the detection unit includes a sample amplification region, and in the sample amplification region, the primer pair according to any one of claims 5-8 or the kit according to claim 8 is provided.

10. The system according to claim 9, wherein, The system further includes an analysis unit, and the analysis unit jointly judges the risk of dental caries occurrence by analyzing the copy numbers of the Smu1335, UA140, and Orf-H subtype genes obtained by the detection unit.

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