KR101735028B1 - Quantitative and qualitative methods of detecting bacteria related to dental caries using Real-time PCR and Melting Curve Analysis - Google Patents

Abstract

Through simultaneous detection of qualitative analysis for four strains of microorganisms of streptococcus genus and four strains of Lactobacillus genus, the present invention can detect the same with high sensitivity and specificity to conduct clinical diagnosis rapidly and easily with high reliability. The present invention comprises the following steps: (a) extracting DNA; (b) amplifying the gene by real-time polymerase chain reaction (PCR); (c) obtaining a melting curve; and (d) detecting Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus.

Description

[0001] The present invention relates to a method for quantitative and qualitative determination of causative bacteria of dental caries using real-time PCR and melting curve analysis,

The present invention relates to a method for quantitative and qualitative examination of causative bacteria for tooth decay using real-time PCR and melting curve analysis. More particularly, the present invention relates to a method for quantitative and qualitative examination of streptococcus (Streptococcus) in Microbiology 4 species and Lactobacillus bacteria (Lactobacillus) in real-time polymerase chain reaction using a EvaGreen fluorescent material with respect to the four kinds of microorganisms (Real-time PCR) and melting curve analysis (melting curve analysis) to perform successively the And a method for simultaneously detecting intermittent quantitation and interspecific qualitative assays of microorganisms.

Tooth decay refers to the damage of tooth enamel due to the acid generated by the decomposition of sugar and starch by bacteria in the mouth. The cause of this is the absence of proper oral hygiene control, Anti-caries Immune reactions, dietary habits, and various structures due to tooth structure are involved, but the root cause is polyunsaturated infections caused by dental caries causing the pH to drop below 5.5. To prevent dental caries, it is necessary to detect the risk factors of dental caries prematurely and to prevent the progress of dental caries through appropriate preventive measures. It is important. Therefore, there is a need to develop a system capable of detecting an efficient and sensitive method for prevention of dental caries, possibility of onset, and prognosis after treatment.

There are more than 300 kinds of bacteria in the human oral cavity. Of these bacteria, Mutans streptococci and Lactobacillus spp. Are known to cause dental caries. In addition, because of differences in the productivity of organic acids depending on the species and serotype of these causative organisms, information on the species and serotype contained in each genus as well as the total amount of the causative organism is more useful for more accurate clinical diagnosis.

Streptococcus mutans, a major cause of dental caries, is classified into serotypes according to the chemical composition of RGPs (serotype-specific rhamnose-glucose polymers) present on the cell surface. Serotype c, serotype e, serotype f and serotype k There are four types. Serotype c is the most common type that accounts for more than 80% of the total mutans found in the dental flora. The other serotypes e, f, and k are not commonly found. Serotype e is about 20% and the other serotype f and k is 5%. Serotype k is found only in Japan. Streptococcus mutans serotypes have been reported to produce specific metabolites and produce immune responses according to each type. Recent studies have reported that infectious endocarditis is caused by serotypes e and f, and serotype f causes hemorrhagic stroke and inflammation related diseases And it has been revealed that the disease is related not only to dental caries, but also to systemic diseases.

To date, the dental caries activity test has been carried out using a selective medium which can be used to selectively measure oral pH or to selectively grow Streptococcus mutans or lactobacillus spp. A method of measuring the amount of bacteria that have been used has been mainly utilized. However, this method can be confirmed by cultivating only one kind per test for the dental caries-inducing bacteria to be confirmed in the oral cavity, and it takes more than 3 days to complete the culture test (labor -ious) to be.

Korean Patent No. 10-1562491 discloses a method for detecting and quantifying microorganisms of the genus Streptococcus and microorganisms of the genus Lactobacillus which are recognized as causative organisms of dental caries. However, this method is only capable of quantifying microorganisms, There is a limitation that qualitative analysis is not possible.

Accordingly, the inventors of the present invention overcome the limitations of only the microorganisms of the genus Streptococcus and Lactobacillus, which are recognized as causative agents of dental caries, in Korean Patent No. 10-1562491, thereby further developing a rapid and accurate quantitative analysis technique As a result of diligent efforts to develop a primer set capable of qualitative analysis of microbial species contained in each genus as well as quantitative analysis of dental caries bacteria, Melting Curve Analysis using a primer set according to the present invention, It is possible to clearly distinguish and detect the species of dental caries causing bacteria. Thus, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f, and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1-8.

Another object of the present invention is to provide a kit for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8.

It is another object of the present invention to provide a method for preparing a pharmaceutical composition comprising: (a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using a primer set of SEQ ID NOS: 1 to 8;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) determining the melting temperature from the melting curve to provide a method for detecting Streptococcus mutans serotype c, serotype e, serotype f, and Streptococcus sobrinus.

Another object of the present invention is to provide a composition for simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracase, Lactobacillus crisparis, and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: 9 to 16 .

Another object of the present invention is to provide a kit for simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracase, Lactobacillus crisparis, and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: 9 to 16 .

It is another object of the present invention to provide a method for preparing a pharmaceutical composition comprising: (a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using primers set forth in SEQ ID NOS: 9 to 16;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) checking the melting temperature from the melting curve to detect Lactobacillus plantarum, Lactobacillus casei or paracasei, Lactobacillus crisparis and Lactobacillus salivarius.

In order to accomplish the above object, the present invention provides a composition for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus subtilis comprising the primer set of SEQ ID NOs: 1 to 8.

In order to accomplish another object of the present invention, there is provided a kit for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8.

According to another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition comprising: (a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using a primer set of SEQ ID NOS: 1 to 8;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) checking the melting temperature from the melting curve to provide Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sbrurus.

In order to accomplish another object of the present invention, the present invention provides a method for detecting simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracasei, Lactobacillus crisparis and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: ≪ / RTI >

In order to accomplish another object of the present invention, the present invention provides a method for detecting simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracasei, Lactobacillus crisparis and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: For example.

According to another aspect of the present invention, there is provided a method for preparing a pharmaceutical composition comprising: (a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using primers set forth in SEQ ID NOS: 9 to 16;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) checking the melting temperature from the melting curve to detect Lactobacillus plantarum, Lactobacillus casei or paracasei, Lactobacillus crisparis and Lactobacillus salivarius.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8.

As used herein, the term " primer " refers to a nucleic acid sequence having a short free 3 'hydroxyl group, which can form a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) for polymerisation and four different dNTPs (deoxynucleoside triphospate) at appropriate buffer solutions and temperatures.

Primers can incorporate additional features that do not alter the primer's basic properties that serve as a starting point for DNA synthesis. In the present invention, the nucleotide sequences of SEQ ID Nos: 1 to 8 and the primers comprising 9 to 16 each include a nucleotide sequence having a sequence homology of 95% or more. For example, when the primer is 20 bp, two or more of the 20 bp errors result in a poorer melting temperature than 5 ° C. However, if only one is wrong, the annealing temperature If you experiment a little down, you will get equal results.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, "capping ", substitution of one or more natural nucleotides into homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents.

In addition, the primer nucleic acid sequences of the present invention may, if necessary, comprise markers detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (e.g., horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., 32P), fluorescent molecules, chemical groups (e.g., biotin) have.

In the present invention, the primers of SEQ ID NOS: 1 to 8 are primers targeting specific gene regions of Streptococcus sp. Microorganisms. More specifically, primer pairs of SEQ ID NOS: 1 and 2 are primer pairs of Streptococcus sibri The primer pair consisting of Streptococcus mutans serotype E, SEQ ID NOs: 5 and 6 is a Streptococcus mutans serotype C and the primer pair consisting of SEQ ID NOs: 7 and 8 is a Streptococcus mutans It is a primer for specifically detecting serotype F.

Preferably, in the present invention, the detection may be by Melting curve analysis. The term "melting" refers to dissociation of double stranded DNA into single stranded DNA. Fusion can be performed by warming up a sample containing double stranded DNA. Melting can be carried out, for example, at 50 캜 to 95 캜, but is not limited thereto.

According to one embodiment of the present invention, the SEQ ID NO: 1 is utilized for to 8 is melting curve analysis primers Streptococcus Streptococcus mutans (Streptococcus mutans) belonging to the (Streptococcus) spp serotype c, serotype e, serotype f And Streptococcus sobrinus were detected only specifically. In particular, when a composition comprising the primer set of SEQ ID NOS: 1 to 8 of the present invention is used, even when the four species of Streptococcus sp. Microorganisms are simultaneously analyzed, since the melt peaks are clearly distinguished, each desired microorganism is clearly detected I can do it.

The present invention also provides a composition, wherein said composition further comprises a primer set of SEQ ID NOs: 17 to 19 and a probe set of SEQ ID NOs: 24 to 25.

The primer sets of SEQ ID NOs: 17 to 19 and the probe sets of SEQ ID NOs: 24 to 25 are primers and probes capable of detecting and quantifying Streptococcus microorganisms among the causative microorganisms causing dental caries. More specifically, the Streptococcus microorganisms are Streptococcus mutans and Streptococcus sobrinus .

When the real-time PCR and the fusion curve analysis are successively performed using the primer set of SEQ ID NOs: 1 to 8, the primer set of SEQ ID NOs: 17 to 19, and the probe set of SEQ ID NOs: 24 to 25, It is possible to quantitatively analyze microorganisms of the genus Streptococcus which are present in the genus Streptococcus, as well as to qualitatively detect microorganisms of the genus Streptococcus simultaneously.

According to one embodiment of the present invention, a composition comprising the primer set of SEQ ID Nos: 1 to 8, the primer set of SEQ ID Nos. 17 to 19 and the probe set of SEQ ID Nos. 24 to 25, It is possible to quantitatively analyze Streptococcus sp. Microorganisms in the target sample, and it is confirmed that accurate qualitative analysis is also possible.

The present invention also provides a kit for simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8.

The kit of the present invention may further comprise a primer set of SEQ ID NOs: 17 to 19 and a probe set of SEQ ID NOs: 24 to 25, in addition to the primers of SEQ ID NOs: 1 to 8. In addition, the components used in the PCR composition can be used, and non-limiting examples of components of such a composition may include reaction buffer solution, dNTP, Mg2 + ion, DNA polymerase.

The buffer solution for reaction may be 1 to 10 mM TrisHCl, 10 to 40 mM KCl (pH 9.0), and the dNTP may be selected from dATP, dTTP, dGTP and dCTP.

The kit may include stabilizers and / or non-reactive dyes for improving experimental convenience, stabilization, and reactivity.

The non-reactive dye material should be selected from a substance which does not affect the polymerase chain reaction, and is intended to be used for analysis or identification using the polymerase chain reaction product. Materials satisfying these conditions may be used as water-soluble dyes such as rhodamine, tamla, lactose, bromophenol blue, xylenecanol, bromocresol red, and cresol red. The non-reactive dye material may be contained in an amount of 0.0001-0.01% by weight based on the total weight of the composition, and is preferably contained in an amount of 0.001-0.005% by weight. When the water-soluble dye is added in an amount of more than 0.01% by weight based on the total weight of the composition, a high concentration of a water-soluble dye may act as a reaction inhibitor in a polymerase chain reaction.

In addition, the polyhydric alcohols may be used as a stabilizing material for stabilizing the kit components of the present invention, and it is preferable to use at least one of glucose, glycerol, mannitol, galactitol, glucitol and sorbitol . The polyhydric alcohol may be contained in an amount of 10 to 500 mM, and preferably in an amount of 50 to 300 mM. If the polyhydric alcohol is more than 500 mM, it is difficult to dissolve in an aqueous solution due to the solubility of the water-soluble polymer itself. Due to the high viscosity, it is difficult to sufficiently mix and the volume becomes larger than necessary. For the polymerase chain reaction, It does not easily dissolve when dissolved. In addition, there is a problem that a high concentration of a water-soluble polymer can act as a reaction inhibitor in a polymerase chain reaction. When the concentration is less than 10 mM, the target enzyme and surface water molecules can not be sufficiently coated and protected. Therefore, it is difficult to expect an effective stabilization effect of the enzyme. Also, since the viscosity of the solution is too low, And there is a problem that the enzyme can not be sufficiently protected. In the present invention, it is possible to use gelatin, small blue albumin, Thesit, PEG-8000 as stabilizers in addition to polyhydric alcohols.

The kit component may be provided in a liquid form and is preferably dried to increase stability, simplicity of storage and long-term storage. The drying can be performed by a known drying method such as general room temperature drying, warm drying, freeze drying, and vacuum drying, and any drying method can be used as long as the composition is not lost.

In the present invention, various DNA polymerases can also be used in the amplification step of the present invention, and include "Clenow" fragments of E. coli DNA polymerase I, thermostable DNA polymerase and bacteriophage T7 DNA polymerase. Preferably, the polymerase is a thermostable DNA polymerase obtainable from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu) . Most of these enzymes can be isolated from the bacteria itself or commercially available. In addition, the polymerase used in the present invention can be obtained from cells expressing high levels of the cloning gene encoding the polymerase.

The present invention also relates to

(a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using a primer set of SEQ ID NOS: 1 to 8;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) checking the melting temperature from the melting curve to provide Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sbrurus.

In the step (a), the clinical sample may be a saliva sample or a tooth sample collected in the oral cavity, and the DNA may be extracted according to a conventional method known in the art (see Sambrook, 9: 242 (1991); Ausubel, FM et al., ≪ RTI ID = 0.0 > , Current Protocols in Molecular Biology, John Willey & Sons (1987), and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)).

In the step (b), PCR reaction is well known in the art, and the target nucleic acid is amplified by repeating the steps of denaturation, annealing and extension using a target nucleic acid as a template and using a primer specific for the target nucleic acid. .

Real-time PCR analysis can be performed using commercially available real-time PCR instruments. For example, SLAN real time PCR detection system (LG Life Sciences, Korea), LightCycler 96 / (Bio-Rad, USA), Rotor-Gene (Corbett, Australia), OpticonTM (PharmaTech, USA), ABC 7500 fast / 7500 / PRISMTM 7000/7700 (Applied Biosystems, USA) And the like, but the present invention is not limited thereto.

In the present invention, the primer set of the step (b) can be used for successfully amplifying the gene of the Streptococcus microorganism by real-time PCR without interference between the respective primers constituting the primer set.

Therefore, when performing the real-time PCR using the primer set, it is very reliable and quick to carry out the microorganism belonging to Streptococcus which is a dental caries inducing microorganism and Streptococcus mutans serotype c ( Streptococcus mutans ) contained in Streptococcus , serotype e, serotype f, and Streptococcus sobrinus .

Since these detections exhibit high specificity and sensitivity, primers set forth in SEQ ID NOS: 1 to 8 capable of detecting microorganisms of the genus Streptococcus . The present invention is based on the inventor's intellectual property right registered as " Only the quantification of Streptococcus sp. And Lactobacillus sp. Microorganisms recognized as causative microorganisms of dental caries in primers and probes for multiplex real-time PCR for simultaneous detection and their use (No. 10-1562491) And the rapid and precise quantitative analysis technique is further developed to confirm not only the quantitative analysis of the causative bacteria of the dental caries but also the qualitative analysis of the major microbial species contained in each genus by using the melting curve analysis method, Clinical diagnosis of carcinogens It is very excellent in the point that (Example 4).

In the present invention, step (c) is a step of melting the amplified amplified product to obtain a melting curve. The term "melting" refers to dissociation of double stranded DNA into single stranded DNA. Fusion can be performed by warming up a sample containing double stranded DNA. Melting can be carried out, for example, at 50 ° C to 95 ° C.

The step of melting the amplified amplification product to obtain a melting curve can be performed using a fluorescent dye. The fluorescent dyes may be, for example, but not limited to SYBR Green, SYTO9, EvaGreen, LC Green, LC Green Plus, Reso Light, or combinations thereof.

The melting curve can be obtained by measuring the fluorescence intensity according to the wavelength depending on the type of dye used. For example, the fluorescence intensity at 510 nm can be measured to obtain the melting curve.

The step (d) is a step of detecting Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus by checking the melting temperature from the melting curve obtained in the step (c).

The melting point (Tm) refers to the temperature at which half of the double helix DNA is separated into individual strands, ie, 50% of the denaturation. The melting temperature may vary depending on the DNA type of the nucleic acid, the polynucleotide sequence, the GC content, the length of the polynucleotide, the presence of the nucleotide variation, and the like.

The present inventors have found that the primer pairs of SEQ ID NOs: 1 and 2, the primer pairs of SEQ ID NOs: 3 and 4, the primer pairs of SEQ ID NOs: 5 and 6, and the primer pairs of SEQ ID NOs: 7 and 8 do not overlap each other And the peaks indicated by the respective primers in the melting curve were designed so as not to overlap with each other, so that four species of Streptococcus sp. Microorganisms could be detected accurately, quickly, and simultaneously.

The present invention further comprises a primer set of SEQ ID NOs: 17 to 19 and a set of probes of SEQ ID NOs: 24 to 25 in the step (b), further comprising the step of quantifying the Streptococcus microorganism in the step (d) The method comprising the steps of:

Regarding the "quantification" in the step (b) of the present invention, the real-time PCR method is classified into absolute quantification and relative quantification. In order to quantify the target DNA in the sample, It is essential to prepare a standard curve of the sample. Quantitative real-time PCR is performed simultaneously on standard and unknown samples to derive a calibration curve from a standard sample and quantify the amount of target DNA in the unknown sample from this calibration curve. At this time, the calibration curve (x axis: concentration of the standard sample, y axis: threshold cycle number) from the standard sample is ideal as the correlation index (R2) approaches 1.

In the present invention, quantification using real-time PCR generally means a series of reactions using PCR to quantify the amount of template DNA at the start of the amplification reaction. Quantitative PCR includes internal standard methods using standard reference standards and competitive methods using molecules competing in amplification reactions. In order to accurately and easily determine the number of molecules of each sequence, a PCR method using a template DNA sequence as a standard called a standard sample is used, and the progress of the reaction, that is, the degree of amplification is checked at any time during the reaction . Therefore, in the present invention, " quantitative " means a PCR for quantifying the amount of template DNA at the start of the amplification reaction, and means a PCR capable of confirming the amplification of a molecule to be amplified at any time during the reaction. For quantification, a combination of such a PCR and a calibration curve associating the number of template DNA molecules at the start of the reaction with a signal that is an index of the amplification is combined. This calibration curve can be prepared using standard samples with a known number of molecules.

In the present invention, quantitative real-time PCR is performed using 16s rRNA of the Streptococcus sp. Microorganism and / or lactobacillus sp. Microorganism to be quantified as a standard sample using a primer for specifically amplifying the gene, (CT) of the target gene present in the sample based on the calibration curve prepared from the amplification of the standard sample, and the concentration of the microorganism of the genus Streptococcus and / or the microorganism of the genus Lactobacillus can be predicted.

Through this method, it is possible not only to quantitatively analyze the Streptococcus microorganisms present in the sample by one experiment, but also to conduct a qualitative analysis at the same time, thereby clearly distinguishing which species of microorganism is contained.

The present invention also provides a composition for simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracase, Lactobacillus crisparis and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: 9 to 16.

The primers of SEQ ID NOS: 9 to 16 are primers targeting a specific gene region of a microorganism belonging to the genus of Lactobacillus . That is, the primer binds specifically to microorganisms of a specific species in the genus Lactobacillus without binding to other microorganisms.

More specifically, the primer pairs of SEQ ID NOs: 9 and 10 are Lactobacillus plantarum, the primer pairs of SEQ ID NOs: 11 and 12 are Lactobacillus casei or paracasei, the primer pairs of SEQ ID NOs: 13 and 14 are Lactobacillus crisparis, The primer pairs of SEQ ID NOs: 15 and 16 are primer pairs that specifically detect Lactobacillus salivarius.

The primer sets of SEQ ID NOS: 9 to 16 specifically detected only four microorganisms belonging to the genus Lactobacillus , and no PCR products were observed for microorganisms belonging to another genus, (Example 1).

The present invention also provides a composition, wherein said composition further comprises a set of primers of SEQ ID NOs: 20 to 23 and a set of probes of SEQ ID NO: 26.

The present invention also provides a kit for simultaneous detection of Lactobacillus plantarum, Lactobacillus casei or paracase, Lactobacillus crisparis and Lactobacillus salivarius comprising the primer set of SEQ ID NOS: 9 to 16.

The present invention also relates to

(a) extracting DNA from a clinical sample;

(b) amplifying the obtained DNA by real-time PCR using primers set forth in SEQ ID NOS: 9 to 16;

(c) melting the amplified amplification product to obtain a melting curve; And

(d) checking the melting temperature from the melting curve to detect Lactobacillus plantarum, Lactobacillus casei or paracasei, Lactobacillus crisparis and Lactobacillus salivarius.

The respective steps are the same as described above.

The method may further comprise the step of (b) further comprising a set of primers set forth in SEQ ID NOS: 20 to 23 and a set of probes set forth in SEQ ID NO: 26, wherein said step (d) further comprises quantitating Lactobacillus sp. The method comprising the steps of:

The above steps are the same as described above.

As described above, only microorganisms of the genus Streptococcus and lactobacillus species, which are causative agents of dental caries, are specifically detected, and each microorganism is detected with excellent sensitivity by Real-time PCR method and Melting curve analysis A primer set that can be used has not been reported so far.

When real-time PCR and Melting curve analysis are performed using the composition and method according to the present invention, microorganisms belonging to the genus Streptococcus which is a causative organism of dental caries and Streptococcus mutans contained in Streptococcus mutans serotype c, serotype e, serotype f, and Streptococcus sobrinus . The microorganism in the genus Lactobacillus and the lactobacillus plantarum contained in the genus Lactobacillus, Lactobacillus casei / Bacillus crispa sp. And Lactobacillus salivarius can be detected simultaneously in a quantitative and qualitative manner, so that clinical diagnosis can be quickly and easily performed with high reliability.

1 is the genomic DNA from 1ng / ul concentration of 10-fold serial dilution by Streptococcus mutans (Streptococcus mutans ATCC25175 ([A] , Cy5; blue) and Lactobacillus Kasei (Lactobacillus Casei ATCC334 ([B] , Cy5; green) (A: S. mutans , B: L. casei ) under real-time PCR conditions.
Figure 2 shows a standard curve of the Streptococcus mutans (Streptococcus mutans ATCC25175 ([A] ) and Lactobacillus Kasei (Lactobacillus Casei ATCC334 ([B] ) (A: S. mutans, B: L.casei).
Fig. 3 is a diagram showing a failure analysis according to a primer pair in the fusion curve analysis method. Fig.
FIG. 4 is a fluorescence graph showing real-time PCR reaction and melting curve analysis reaction in a standard strain using the composition of the present invention (A: analysis of standard strains of four strains of Streptococcus species, B: Analysis of Four Standard Species Fusion Curve.
FIG. 5 is a fluorescence graph for a real-time PCR reaction and a melting curve analysis reaction in clinical sample 1 using the composition of the present invention (A: Real-time PCR reaction, B: Melting curve analysis reaction, 5B, microorganisms of the genus Streptococcus were detected on the left side and microorganisms of the genus Lactobacillus were detected on the right side.
FIG. 6 is a fluorescence graph for real-time PCR reaction and melting curve analysis reaction in clinical sample 2 using the composition of the present invention (A: Real-time PCR reaction, B: Melting curve analysis reaction, 6B, the left side shows detection of Streptococcus sp. Microorganisms, and the right side shows detection of microorganisms in Lactobacillus sp.).
FIG. 7 is a fluorescence graph for a real-time PCR reaction and a fusion curve analysis reaction in clinical sample 3 using the composition of the present invention (A: Real-time PCR reaction, B: Melting curve analysis reaction, 7B, the left side shows detection of Streptococcus sp. Microorganisms, and the right side shows detection of Lactobacillus sp. Microorganisms).

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Preparation of experiment>

1. Preparation of primer and probe

The primers and probes used in the present invention are obtained by analyzing the Genebank sequence registered in the gene bank (www.ncbi.nlm.nih.gov) operated by NCBI under the National Institute of Health, using DNAsis program of Hitachi Software Co., The nucleotide sequence was determined and analyzed again with BLAST (www.ncbi.nlm.nih.gov/BLAST/) to confirm that it is a primer base sequence capable of amplifying only Streptococcus or Lactobacillus sp. Microorganisms.

The primers and probes used in the present invention can be obtained by secreting a specific gene of Streptococcus sp. Or Lactobacillus sp. Microorganism in the entrez of Genebank, analyzing the conserved region using the clustal W2 multi-nucleotide sequence analysis program, The sequence was determined and analyzed by BLAST (www.ncbi.nlm.nih.gov/BLAST/) to confirm that it is a primer base sequence capable of amplifying only Streptococcus or Lactobacillus microorganisms.

2. Synthesis of primer

The primers analyzed in Example 1 were synthesized using methods such as oligonucleotide synthesis described in 10.42 of molecular cloning 3rd edition (sambrook and rusell, cold spring harborlaboratory press, New York, USA, 2001) ).

3. Extraction of DNA from microorganisms

Genomic DNA was extracted from the standard strain using the Exgene ^TM Cell SV DNA Extraction Kit (Jinol Biotechnology, Korea) according to the manufacturer's instructions. The extracted genomic DNA was measured using NanoDrop 2000 (Thermo Scientific co., USA) spectrophotometer and diluted to 10 pg / ul.

&Lt; Example 1 >

Identification of primer specificity

In order to confirm the specificity of the primers of SEQ ID NOS: 1 to 8 prepared in the above preparation step for Streptococcus mutans serotype C, serotype E, serotype F and Streptococcus sbrinus microorganisms contained in Streptococcus sp. , The following experiments were carried out to confirm the specificity of the primers of SEQ ID NOS: 9 to 16 against Lactobacillus plantarum, Lactobacillus casei / Paracasei, Lactobacillus crisparis, and Lactobacillus salivarius microorganisms contained in Lactobacillus .

Primers for Detection of Streptococcus sp. Microorganisms (SEQ ID NOS: 1 to 8) Specificity The PCR reaction solutions used for the assay were 2ul (1ng / ul) of template DNA, 0.5ul (10 x concentration containing 15 mM MgCl ₂ solution) of each primer of SEQ ID Nos. 1 to 8, 0.4 uL (1 unit) of Solg ^TM h-Taq DNA Polymerase (Solgent co., Korea) (0.1 unit) of SolGent ^™ Uracil-DNA Glycosylase (Solgent co., Korea), 1 ul (dATP, dCTP, dGTP, 2 mM each) and dUTP DNase and RNase free water (Sigma-Aldrich co., USA) were added to the mixture of dNTP (Solgent co., Korea).

The PCR reaction solution used to verify the specificity of the Lactobacillus sp. Microorganism detection primers (SEQ ID NOS: 9 to 16) contained 2 ul (1 ng / ul) of template DNA, 0.5 ul (10 pmol / primer, 0.4ul (1 unit) of ^{Solg TM h-Taq DNA Polymerase (} Solgent co., Korea), 3ul h-Taq reaction buffer of (15 mM MgCl ₂ solution containing a concentration 10X) (Solgent co., Korea) (Solgent co., Korea) solution containing 0.1 μl (0.1 unit) of SolGent ^™ Uracil-DNA Glycosylase (Solgent co., Korea) and 1 μl of each of dATP, dCTP and dGTP DNase, and RNase free water (Sigma-Aldrich co., USA) were added to make 30ul of the total reaction solution.

The PCR reaction was carried out by using a Peltier thermal cycler (Model PTC-100 DNA Engine ^™ , MJ Research Inc., USA) for 5 minutes at 50 ° C. for 10 min. denaturation and hot start Taq were performed, followed by denaturation at 94 for 20 seconds, annealing and extension for 72 to 40 seconds, and then a post-extension of 72 for 5 minutes . Amplified PCR products were amplified and amplified using Mupid-2plus electrophoresis (Mupid, Japan).

The results are shown in Table 5 below.

As shown in the following Table 5, the primer sets of SEQ ID NOS: 1 to 8 capable of specifically detecting Streptococcus sp. Microorganisms were prepared for each of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus And only specific microorganisms were detected. As a result, it was confirmed that the microorganisms including Lactobacillus sp.

In addition, primer sets of SEQ ID NOS: 9 to 16 capable of specifically detecting Lactobacillus sp. Microorganisms include microorganisms such as Lactobacillus plantarum, Lactobacillus casei / Paracasei, Lactobacillus crisparis, and Lactobacillus salivarius, And the microorganisms belonging to other genera showed no reaction. Thus, it was confirmed that the specificity was excellent.

Identify the specificity of the designed primer set Bacterial strains Primer set SEQ ID NO: 17 to 19 SEQ ID NOS: 1 and 2 SEQ ID NOS: 3 and 4 SEQ ID NOS: 5 and 6 SEQ ID NOS: 7 and 8 SEQ ID NOs: 20 to 23 SEQ ID NOS: 9 and 10 SEQ ID NOS: 11 and 12 SEQ ID NOS: 13 and 14 SEQ ID NOS: 15 and 16 Streptococcus mutans serotype c + - - + - - - - - - Streptococcus mutans serotype e + - + - - - - - - - Streptococcus mutans serotope f + - - - + - - - - - Streptococcus sobrinus ATCC 33478 + + - - - - - - - - Streptococcus mitis KCOM1379 - - - - - - - - - - Streptococcus gordonii ATCC10558 - - - - - - - - - - Streptococcus oralis ATCC35037 - - - - - - - - - - Streptococcus sanguinis KCOM1017 - - - - - - - - - - Streptococcus parasanguinis ATCC15912 - - - - - - - - - - Streptococcus salivarius ATCC7073 - - - - - - - - - - Streptococcus thermophilus ATCC19258 - - - - - - - - - - Streptococcus vestibularis ATCC 49124 - - - - - - - - - - Lactobacillus casei ATCC334 - - - - - + - + - - Lactobacillus paracasei ATCC335 - - - - - + - + - - Lactobacillus fermentum ATCC11739 - - - - - + - - - - Lactobacillus acidophilus ATCC4356 - - - - - + - - - - Lactobacillus gasseri ATCC 33323 - - - - - + - - - - Lactobacillus crispatus ATCC33820 - - - - - + - - + - Lactobacillus rhamnosus ATCC7469 - - - - - + - - - - Lactobacillus plantarum ATCC10241 - - - - - + + - - - Lactobacillus salivarius ATCC11741 - - - - - + - - - + Campylobacter rectus ATCC33238 - - - - - - - - - - Eikenella corrodens ATCC23843 - - - - - - - - - - Eubacterium nodatum ATCC33099 - - - - - - - - - - porphyromonas gingivalis ATCC33277 - - - - - - - - - - Prevotella nigrescens ATCC33563 - - - - - - - - - - Tannerella forsythia ATCC43037 - - - - - - - - - - Treponema denticola ATCC35405 - - - - - - - - - - Fusobacterium nucleatum ATCC25586 - - - - - - - - - - Prevotella intermedia ATCC25611 - - - - - - - - - - Esherichia coli K12 - - - - - - - - - -

As can be seen from the above table, the primer sets of SEQ ID NOs: 17 to 19, which are primers for quantitation of Streptococcus sp. Microorganisms, are characterized in that Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus, , And the effect

The primer sets of SEQ ID NOs: 20 to 23, which are primers for the quantification of microorganisms of the genus Lactobacillus, are used to qualitatively distinguish between Lactobacillus plantarum, Lactobacillus casei / Paracasei, Lactobacillus crisparis and Lactobacillus salivarius .

In contrast, the primer pairs of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8 showed accurate detection of the desired species in Streptococcus sp.

The primer pairs of SEQ ID NOs: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16 respectively showed the effect of accurately detecting the desired species in the genus Lactobacillus.

On the basis of these results,

(i) the primers of SEQ ID NOS: 1 to 8 were mixed to determine whether four species of Streptococcus sp. microorganisms can be distinguished accurately and rapidly and simultaneously,

(ii) when the primer sets of SEQ ID NOS: 17 to 19, the probe sets of SEQ ID NOS: 24 to 25, and the primer sets of the SEQ ID NOS: 1 to 8 are mixed, each of the primers does not exhibit interference effect with each other and the microorganism of Streptococcus Were quantitatively detected and qualitatively distinguished.

In addition,

(i) the primers of SEQ ID NOS: 9 to 16 were mixed to determine whether four species of Lactobacillus sp. microorganisms could be distinguished accurately and rapidly and simultaneously,

(ii) when the primers set forth in SEQ ID NOs: 20 to 23, the primers set forth in SEQ ID NO: 26, and the primer sets set forth in SEQ ID NOS: 9 to 16 are mixed, the respective primers do not exhibit mutual interference effects and the Lactobacillus microorganisms are quantitatively And to judge whether or not they can be distinguished qualitatively simultaneously with detection.

&Lt; Example 2 >

Standard curve creation and detection sensitivity evaluation

The present inventors quantitatively analyzed the Streptococcus sp. Microorganisms and the Lactobacillus sp. Microorganisms causing dental caries in a composition comprising the primer set of SEQ ID NOs: 17 to 23 and the probe set of SEQ ID NOs: 24 to 26 in Korean Patent No. 10-1562491 I have confirmed that I can do it.

Thus, the present inventors have found that when the primer set for quantitative detection and the probe set are mixed with the primer set of SEQ ID NOs: 1 to 8 or the primer set of SEQ ID NOs: 9 to 16, the Streptococcus sp. Microorganism or the Lactobacillus sp. Microorganism can be quantitatively detected And to determine whether or not it can be detected by the detection sensitivity.

In order to determine the detection sensitivity of Real-time PCR Streptococcus mutans (Streptococcus mutans) ATCC25175 and Lactobacillus Kasei (Lactobacillus Casei) by targeting ATCC334, after the extraction of the genome DNA, 10-fold fluorescence intended for serial dilution samples The amplification curve was confirmed (Fig. 1), and a standard graph was created based on the CT value of the sample shown in the result of the fluorescence amplification curve (Fig. 2). The genome size of each standard strain was confirmed in the NCBI database (http://ncbi.nlm.nih.gov) and based on the number of Avogadro (6.02 × 10 ²³ molecules per mole) The genomic DNA copy number for the strain was calculated and converted to a log value for drawing a standard graph as shown in Table 6 below.

In order to determine whether the quantitative and qualitative determination of the Streptococcus sp. Can be simultaneously confirmed, the reaction solution of the real-time PCR was subjected to PCR using 2ul (1ng / ul) of template DNA, 0.5ul (10 pmol / And Proud of SEQ ID NOs: 24 and 25, 0.1ul of 20X EvaGreen (Biofact, Korea), 0.4ul (1 unit) of Solg ^TM h-Taq Taq reaction buffer (Solgent co., Korea), 3 μl (10 × concentration containing 15 mM MgCl ₂ solution) and 0.1 μl (0.1 unit) SolGent ^TM DNase, RNase free water (Sigma-Aldrich co., USA) was added to dNTP (Solgent co., Korea) mixture of Uracil-DNA Glycosylase (Solgent co., Korea) and 1ul (dATP, dCTP, dGTP 2 mM and dUTP 6 mM) Was added so that the total reaction solution became 30ul.

Similarly, 2 μl (1 ng / μl) of template DNA, 0.5 μl (10 pmol / μl) of primers of base sequence 9 to 16 and primers of SEQ ID Nos. 20 to 23 and 0.1 μl of the probe, 0.3ul of SEQ ID NO: 26 of the 10 pmol/ul) 20X EvaGreen (bio fact, Korea), 0.4ul (1 unit ) Solg TM h-Taq DNA Polymerase (Solgent co in., Korea), 3ul (15 mM MgCl a 10X concentration contains the _second solution) in the h-Taq reaction buffer (Solgent co ., Korea), SolGent TM Uracil-DNA Glycosylase (Solgent co of 0.1ul (0.1 unit) to prevent contamination., Korea), 1ul (dATP DNase, RNase free water (Sigma-Aldrich co., USA) was added to dNTP (Solgent co., Korea) solution of dCTP, dGTP 2 mM and dUTP 6 mM respectively. The PCR reaction was carried out by using a Real-time PCR Thermal Cycler (Model CFX96 Real-time PCR Detection System, Bio-Rad Inc., USA) After the initial denaturation and hot start Taq activation, denaturation at 94 for 20 sec and annealing and extension at 72 sec for 40 sec were repeated 40 times. Melting curve analysis was performed to measure the fluorescence value by increasing 0.2 to 65 from 95 to 95 for the melting curve analysis. Fluorescence values were measured at 72 and data were analyzed using Bio-Rad CFX Manager 2.1. The cycle threshold (Ct) starts with a logarithmic amplifier with a known number of DNA concentrations and the number of bacteria per sample is measured by the standard curve for the strain.

With the results as described above, by Real-time PCR are designed to the invention Streptococcus mutans (Streptococcus mutans) ATCC25175 and Lactobacillus Kasei (Lactobacillus Casei) the genome DNA of ATCC334 can be detected in a longitudinal specific 100fg / up ul . In addition, considering the fact that the genomic DNA sizes of the standard strains were 2.03 Mbp and 2.92 Mbp, respectively, the detection sensitivities according to the present invention were 45.7 copy / ul and 31.8 copy / ul, respectively. When the probe set of SEQ ID Nos. 24 to 26 is mixed with the primer set of SEQ ID Nos. 1 to 8 or the primer sets of SEQ ID Nos. 9 to 16, it is possible to detect the Streptococcus sp. Microorganism or the Lactobacillus sp. Microorganism with excellent sensitivity it means

&Lt; Example 3 >

Assessment of Standard Bacterial Qualification by Melting Curve Analysis

The composition of the present invention in which the composition of the present invention is mixed with the primer set of SEQ ID Nos: 1 to 8 or the primer set of SEQ ID NOS: 9 to 16 is subjected to Melting curve analysis using Real-time PCR and EvaGreen However,

(i) the composition of the present invention in which the primer sets of SEQ ID NOS: 1 to 8 are mixed, accurately and quickly and simultaneously detects Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus Whether you can, or

(ii) the primer sets of SEQ ID NOS: 9 to 16 can accurately and rapidly and simultaneously detect Lactobacillus plantarum, Lactobacillus casei / Paracase, Lactobacillus crisparis, and Lactobacillus salivarius, respectively , The same composition as that of the Real-time PCR reaction solution of Example 2 was used, and the DNA concentration of the standard strain was adjusted to 100 pg / ul. When evaluating causative organisms of Streptococcus spp. And Lactobacillus spp. In order to confirm the caries (dental caries) activity in the clinical analysis experiment, the activity classification criteria were 10 ¹ / ul of low concentration and 10 ² / ul and 10 ³ / concentration, high concentration is of 10 ³ / ul or higher density in consideration of the point that is no less than 10 ⁴ / ul, as shown in the respective type strain is assumed that the presence in the sample to the maximum clinically Table 7 100 pg / ul. &lt; / RTI &gt; This is to confirm whether or not each of the standard strains can clearly distinguish each qualitative analysis result even if there is a clinically corresponding maximum concentration in the reaction solution of the present invention.

The DNA of the standard strain includes (i) DNA of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus to determine the effect of the primer set of SEQ ID NOS: 1 to 8, or ii) DNA of Lactobacillus plantarum, Lactobacillus casei / Paracasei, Lactobacillus crisparis and Lactobacillus salivarius is included to determine the effect of the primer set of SEQ ID NOS: 9 to 16.

The object of the present invention is to provide a method for detecting (i) four strains of Streptococcus sp. Microorganisms using the primer set of SEQ ID NOS: 1 to 8, precisely, promptly, (Ii) four kinds of Lactobacillus sp. Microorganisms are accurately, rapidly, and simultaneously detected using the primer set of SEQ ID NOS: 9 to 16, so that in this embodiment, the primer sets of the present invention are used Melting curve analysis was performed to determine whether the detection peaks corresponding to the respective microorganism species can be clearly distinguished from each other to enable accurate discrimination.

Melting Curve Analysis A primer for qualitative analysis should show one Melting peak in one primer. Melting Curve Analysis When several sets of primers for qualitative analysis are simultaneously tested in one reaction, And the Melt Peak values are clearly distinguishable. Even if a pair of primers for detecting the same microorganism is used, the Melt point differs depending on whether any part of the DNA base sequence of the microorganism is selected to prepare a primer having a complementary sequence, the base sequence length of the prepared primer, The shape of the shape also has completely different results. That is, when the primer for melting curve analysis is made incorrectly, two or more peaks may appear in one primer, and when the reaction is proceeded by mixing various kinds of primer pairs as in the present invention, the melt peak So that it is difficult to distinguish each other from each other.

More specifically, an example of a primer made by one of the present inventors is shown in FIG.

The results shown in FIG. 3A show that there are nonspecific Melt peaks in one primer pair, and two peaks are observed even though the reaction proceeds for the purpose of detecting one microorganism.

The results shown in FIG. 3B show the result of the reaction to detect two kinds of microorganisms by mixing two kinds of primer pairs. Since the peak of each primer pair is not distinguishable, one Melt peak . That is, in the case of such a primer pair mixture, it is impossible to qualitatively detect two kinds of microorganisms at the same time.

The results shown in FIG. 3C show the results when the peak of the peak is distinguished by the different primer pairs but the interference pattern is interfered with each other and the peak shape is not correct or the peak position of the melt peak is not clear. In this case, too, it is impossible to simultaneously detect several kinds of microorganisms.

Therefore,

(i) the primer set (for the analysis of Streptococcus sp. microorganisms), the primer set (for quantitation of Streptococcus sp. microorganisms) of SEQ ID NOs: 17 to 19, and the probe set of SEQ ID NOs: 24 to 25 (For quantitative analysis of Streptococcus sp. Microorganisms) were used to detect standard samples. As a result, as shown in the following Table 7, the primer pair set of SEQ ID NOS: 1 to 8 showed the respective Melt peak temperature As shown in FIG. 4A, it was confirmed that each microorganism can be accurately detected even in the melt peak graph.

(ii) a primer set of SEQ ID NOS: 9 to 16 (for lactobacillus microorganism analysis), a primer set of SEQ ID NOS: 20 to 23 (for lactobacilli genus microanalysis) and a probe set of SEQ ID NO: 26 As shown in the following Table 7, the primer pairs set forth in SEQ ID NOS: 9 to 16 showed that the respective Melt peak temperatures for detecting microorganisms did not overlap with each other And as shown in FIG. 4B, it was confirmed that each microorganism can be precisely detected and detected even in the melt peak graph.

(For reference, peaks detected at the rightmost side in the graphs of FIGS. 4A and 4B are Melt peaks of the primers for quantitative analysis, which are independent of the qualitative analysis according to the present invention.)

That is, although the primer sets according to the present invention have four pairs of primer pairs, the desired microorganisms can be accurately discriminated by the melting curve analysis method. In addition, this effect is excellent in that accurate quantitative analysis is possible without interfering with the primer and probe set for quantitative analysis, even though they are mixed together.

<Example 4>

Evaluation of clinical sample application of real-time PCR and Melting curve analysis

The composition of the present invention comprising the primer set of SEQ ID Nos. 1 to 8 or the primer set of SEQ ID Nos. 9 to 16 was used to perform real-time PCR and Melting curve analysis using EvaGreen fluorescent material The following experiment was carried out to confirm whether the target microorganisms were precisely classified and qualitatively analyzed in the clinical sample, and at the same time, the primer and the probe for quantitative analysis exerted the effect.

The same method as in Example 3 was used, but the analysis samples were changed to three kinds of clinical samples. Clinical samples were prepared by using a saliva sample spatially spatially resurfaced by a clinical applicant for 20-30 seconds using a gaggle solution to collect the same saliva as the sample used in the causative bacteria test method to confirm the cavity (dental caries) activity. We used the sample received with the analysis request form and the clinical applicant 's own genetic test consent.

The results are shown in Figs. 5 to 7 and Table 8 below.

As shown in Figs. 5 to 7 and Table 8, qualitative analysis of Streptococcus spp. And four major species contained in Streptococcus spp. Were also possible in clinical samples. Quantitative determination of Lactobacillus spp. And major 4 Qualitative analysis of species was possible.

When the real-time PCR and the Melting Curve Analysis are continuously performed using the test method according to the present invention, the total quantitative analysis of Streptococcus in the dental caries causative organism, the Streptococcus mutans serotype c, serotype e, serotype f and Sterptococcus sobrinus , and the total quantitative analysis of Lactobacillus in the dental caries causing dental caries and Lactobacillus plantarum , Lactobacillus casei, ( Lactobacillus casei / paracasei ), Lactobacillus crispatus ( Lactobacillus crispatus ) and Lactobacillus salivarius ( Lactobacillus salivarius ) can be detected with high sensitivity and specificity, It is possible to make clinical diagnosis quickly and easily with high reliability, The possibilities are very good.

<110> KIM, sung hyun <120> Quantitative and qualitative methods of detecting bacteria          related to dental caries using Real-time PCR and Melting Curve          Analysis <130> NP16-0055 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus sobrinus specific primer forward <400> 1 tcaagatccc gctgaacaaa c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus sobrinus specific primer reverse <400> 2 ggttgcatct tgagcgtatt tc 22 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype E specific primer forward <400> 3 cttaaaaatt ggatttcttt gaaacctcg 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype E specific primer reverse <400> 4 ggtgtttgaa ctaaccattc atctgt 26 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype C specific primer forward <400> 5 ggagcatttg gtgtttcttt attagg 26 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype C specific primer reverse <400> 6 caatctcttg cgagagacga c 21 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype F specific primer forward <400> 7 gtcttatata tttagtggta ggttcttttc tc 32 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus mutans serotype F specific primer reverse <400> 8 gtctatcatc agataatgcc tcagc 25 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum specific primer forward <400> 9 aaatgttccg taagacactt gact 24 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus plantarum specific primer reverse <400> 10 agtttggatc gaacctggct ta 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus casei / paracasei specific primer forward <400> 11 ccgtgagcat atcttgttgg c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus casei / paracasei specific primer reverse <400> 12 cgtcaaccaa gtctgtcttg t 21 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus crispatus specific primer forward <400> 13 cttctttagc acaatcacac gaag 24 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus crispatus specific primer reverse <400> 14 tgtttggctt aacattaaca gtagc 25 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus salivarius specific primer forward <400> 15 tgatatagga tgtacaatcg ctaatacc 28 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus salivarius specific primer reverse <400> 16 tgttttatac ctagttggtt acatttctg 29 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus specific primer forward <400> 17 gcatcactag tagatggacc tgcgtt 26 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus specific primer reverse 1 <400> 18 agcacactat ggttgagcca tagc 24 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus specific primer reverse 2 <400> 19 agcatacatt ggttgagcca atgc 24 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific primer forward <400> 20 gtgttggrrg gtttccgcc 19 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific primer reverse 1 <400> 21 gcaccacctg tcattttgcc c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific primer reverse 2 <400> 22 gcaccacctg tcttagcgtc c 21 <210> 23 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific primer reverse 3 <400> 23 aagacctggt aaggttcttc gcgta 25 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus specific probe 1 <400> 24 aagaacgtgt gtgagagtgg aaagttcaca 30 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus specific probe 2 <400> 25 aagaacgtgt gtaagagtgg aaagcttaca ca 32 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus specific probe <400> 26 ctcaaaggaa ttgacggggg cc 22

Claims (12)

A composition for qualitative simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8.
The composition of claim 1, wherein the detection is by Melting curve analysis.
The composition of claim 1, wherein the composition further comprises a set of primers of SEQ ID NOs: 17 to 19 and a set of probes of SEQ ID NOs: 24 to 25.
A kit for qualitative simultaneous detection of Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus comprising the primer set of SEQ ID NOS: 1 to 8;
(a) extracting DNA from a clinical sample;
(b) amplifying the obtained DNA by real-time PCR using a primer set of SEQ ID NOS: 1 to 8;
(c) melting the amplified amplification product to obtain a melting curve; And
(d) Determining the melting temperature from the melting curve to qualitatively distinguish Streptococcus mutans serotype c, serotype e, serotype f and Streptococcus sobrinus.
The method according to claim 5, wherein the step (b) further comprises the primer set of SEQ ID NOs: 17 to 19 and the probe set of SEQ ID NOs: 24 to 25, wherein the step (d) comprises quantitating the Streptococcus microorganism &Lt; / RTI &gt;
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