KR102215852B1 - Primer set for detection of periodontal disease associated bacteria and using thereof - Google Patents

Abstract

The present invention relates to a primer for the detection of periodontal disease causative bacteria, and uses thereof. More particularly, the present invention relates to: a primer for the detection of periodontal disease causative bacteria, capable of qualitatively and quantitatively detecting periodontal disease causative bacteria and capable of detecting periodontal disease causative bacteria having excellent species specificity; a composition and a kit for detecting periodontal disease causative bacteria; and a method for detecting periodontal disease causative bacteria using the same.

Description

Primer set for detection of periodontal disease-causing bacteria and its use {PRIMER SET FOR DETECTION OF PERIODONTAL DISEASE ASSOCIATED BACTERIA AND USING THEREOF}

The present invention relates to a primer set for detecting periodontal disease causative bacteria and uses thereof, and more specifically, periodontal disease capable of qualitatively and quantitatively detecting periodontal disease-related causative bacteria, and capable of detecting periodontal disease-related causative organisms with excellent species specificity It relates to a primer for detection of causative bacteria, a composition for detection, a kit, and a method for detecting periodontal disease causative bacteria using the same.

It is known that there are about 700 bacterial species in the human oral cavity, and numerous bacteria inhabit in lumps to form a dental plaque or dental biofilm. The tooth-to-tooth or between teeth and gums remains without being removed even by brushing, and bacteria proliferate within the tooth-to-earth bacterial film remaining in these areas and act directly on the gingival tissue to induce an immune response or cytotoxicity. It releases substances, causing inflammation of the gingival tissue. This inflammation proceeds to the alveolar bone and causes periodontitis.

Periodontitis is a chronic disease in which alveolar bone is absorbed, which surrounds the roots of gardenia due to a variety of complex oral bacteria being infected in the oral cavity.More than 2/3 of adults around the world are affected, as well as a serious cause of tooth extraction in adults. It is a global disease. In addition, complex bacteria or toxic substances induced therein have a decisive influence in the process of causing or intensifying serious systemic diseases through the bloodstream.

As the aging society enters, the number of elderly patients among these periodontal disease patients is increasing, and periodontal patients with chronic wasting diseases also rapidly increase, resulting in various systemic diseases (e.g., cardiovascular disease, diabetes, low weight preterm birth, rheumatism). Arthritis, chronic kidney disease, upper respiratory pulmonary disease, cancer, memory loss, etc.) have been studied.

The major causative species of periodontal disease are Aggregatibacter actinomycetemcomitans, Pophyromonas gingivalis, Tannerella forsythia, and Treponema denticola ( Treponema denticola), Prevotella intermedia, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, and the like are known. Research to identify other causative species is ongoing. In addition, there are studies showing that the bacterial species associated with periodontal disease differ depending on the area where you live. Therefore, in order to identify the causative agent of periodontal disease, a large-scale epidemiological study targeting all humans is required, and at this time, a detection method with excellent sensitivity and specificity is required while all researchers can use it jointly.

The conventional method of diagnosing periodontal disease uses a periodontal probe that causes considerable pain to the patient.Since this pain is reduced by simple sample collection, precise genetic quantitative analysis of the amount of periodontal disease-causing microorganisms that cause periodontal disease By diagnosing through the method, there is a need to develop a method that can identify the cause of the disease, enable fundamental treatment, and confirm whether periodontal disease is improved during or after treatment.

In addition, the existing microbial testing methods, which are frequently performed in dentistry, do not properly distinguish the type and number of microbes, and the doctor has difficulty in accurately determining and treating pathogenic risk factors based on visual evidence.

Molecular epidemiologic studies to find the causative bacteria related to periodontal disease so far have mostly been qualitatively analyzed using the PCR method that produced primers based on the base sequence of 16s rDNA [G. Conrads et al. "The use of a 16S rDNA directed PCR for the detection of endodontopathogenic bacteria." Journal of Endodontics 23.7 (1997):433-438]. The 16s rDNA sequence has the advantage of having a high positivity rate because it is a location where numerous studies have been carried out, and it has few mutations, but it has a sequence structure similar to that of several microbial species, so it is necessary to optimize the PCR conditions and reaction solution conditions. It is highly likely that non-specific amplification reactions against similar microorganisms will occur.

Therefore, in order to compensate for the above-described problems, the present inventors have conducted an epidemiological study to identify the causative agent of periodontal disease, and in particular, an efficient and highly sensitive method of the causative agent of periodontal disease in order to determine the prognosis after prevention, possibility of onset, and treatment of periodontal disease. Recognizing that the development of a system that can be detected as is urgent, the present invention has been completed.

Korean Registered Patent Publication No. 10-2084862 Korean Patent Application Publication No. 10-2010-0074455

An object of the present invention is to provide a primer for detecting periodontal disease-causing bacteria, a composition for detection, a detection kit, and a method for detecting periodontal disease-causing bacteria using the same.

The technical problems to be achieved by the invention are not limited to the technical problems mentioned above, and other technical problems that are not mentioned can be clearly understood by those of ordinary skill in the art from the description of the present invention.

In order to achieve the above object, the present invention provides a primer for detecting periodontal disease causative bacteria, a detection composition, a detection kit, and a method for detecting periodontal disease causative bacteria using the same.

Hereinafter, the present specification will be described in more detail.

The present invention comprises a primer pair for detection of Campylobacter rectus consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A pair of primers for detecting capnocytophaga sputigena consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; A pair of primers for detection of Eikenella corrodens consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; A pair of primers for detection of Fusobacterium nucleatum consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; A pair of primers for detection of Fusobacterium periodonticum consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10; A pair of primers for detection of Haemophilus parainfluenzae consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; A primer pair for detection of Kingella oralis consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14; A pair of primers for detection of Porphyromonas endodontalis consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16; A pair of primers for detection of Porphyromonas gingivalis consisting of a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18; Prevotella intermedia detection primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20; Prevotella melaniogenica detection primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22; Rothia dentocariosa detection primer pair consisting of a forward primer of SEQ ID NO: 23 and a reverse primer of SEQ ID NO: 24; A pair of primers for detection of Selenomonas noxia consisting of a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26; Streptococcus salivarius detection primer pair consisting of a forward primer of SEQ ID NO: 27 and a reverse primer of SEQ ID NO: 28; Streptococcus sanguinis detection primer pair consisting of a forward primer of SEQ ID NO: 29 and a reverse primer of SEQ ID NO: 30; And a primer pair for detection of Tannerella forsythia consisting of a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32;; For detection of periodontal disease causative bacteria comprising at least one primer pair selected from the group consisting of A primer set is provided.

In the present invention, the periodontal disease is characterized in that it is gingivitis, periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy.

In the present invention, the alveolar bone dysplasia is characterized in that alveolar bone osteoporosis, alveolarbone osteomalacia, or alveolar bone osteopenia.

In addition, the present invention provides a composition for detecting periodontal disease causative bacteria comprising the primer set.

In the present invention, the composition is a polymerase chain reaction (PCR), a real-time polymerase chain reaction (real-time PCR), and a multiplex polymerase chain reaction, Multiplex PCR) is characterized in that it is used in one or more reactions selected from the group consisting of.

In addition, the present invention provides a kit for detecting periodontal disease causative bacteria comprising the primer set.

In the present invention, the kit is a polymerase chain reaction (PCR), a real-time polymerase chain reaction (real-time PCR), or a multiplex polymerase chain reaction, It is characterized in that it is a kit for multiplex PCR).

In addition, the present invention (S1) separating the DNA from the biological sample; (S2) amplifying a target sequence by treating the isolated DNA with the primer set; And (S3) identifying the amplification product; it provides a method for detecting periodontal disease causative bacteria.

In the present invention, the detection method is a polymerase chain reaction (PCR), a real-time polymerase chain reaction (real-time PCR), and a multiplex polymerase chain reaction. , Multiplex PCR), characterized in that at least one selected from the group consisting of.

All matters mentioned in the above primers for detecting periodontal disease causative bacteria, detection compositions, kits, and methods of detecting periodontal disease causative bacteria using the same are applied equally unless contradictory.

The primer for the detection of periodontal disease causative bacteria, the detection composition, kit, and the detection method of periodontal disease causative bacteria using the same according to the present invention have high sensitivity and excellent species specificity, and whether various bacteria have infected periodontal tissues in the oral cavity. Alternatively, the presence or absence of oral bacterial species can be detected and detected quickly and simply, and the causative bacteria that cause periodontal disease can be qualitatively and quantitatively detected. Furthermore, it can be used as a tool to identify bacteriological prognosis evaluation through comparative analysis of the results of bacterial species detection before and after periodontal disease treatment.

The effects of the present invention are not limited to the effects mentioned above, and other effects that are not mentioned will be clearly understood by those skilled in the art from the description of the claims.

1 is a graph showing fluorescence amplification curves shown in real-time PCR experimental conditions of SEQ ID NOs: 1 to 8 of the present invention.
2 is a graph showing fluorescence amplification curves shown in real-time PCR experimental conditions of SEQ ID NOs: 9 to 16 of the present invention.
3 is a graph showing fluorescence amplification curves shown in real-time PCR experimental conditions of SEQ ID NOs: 19 to 24 of the present invention.
4 is a graph showing fluorescence amplification curves shown in real-time PCR experimental conditions of SEQ ID NOs: 25 to 32 of the present invention.

Terms used in the present specification have selected general terms that are currently widely used as possible while considering functions in the present invention, but this may vary depending on the intention or precedent of a technician working in the field, the emergence of new technologies, and the like. In addition, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning of the terms will be described in detail in the description of the corresponding invention. Therefore, the terms used in the present invention should be defined based on the meaning of the term and the overall contents of the present invention, not a simple name of the term.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

The numerical range includes the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were expressly written. All minimum numerical limits given throughout this specification are inclusive of all higher numerical limits as if the higher numerical limits were expressly written. All numerical limits given throughout this specification will include all better numerical ranges within the wider numerical range, as if the narrower numerical limits were expressly written.

Hereinafter, examples of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.

Primer set for detecting periodontal disease causative bacteria

The present invention comprises a primer pair for detection of Campylobacter rectus consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A pair of primers for detecting capnocytophaga sputigena consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; A pair of primers for detection of Eikenella corrodens consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6; A pair of primers for detection of Fusobacterium nucleatum consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8; A pair of primers for detection of Fusobacterium periodonticum consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10; A pair of primers for detection of Haemophilus parainfluenzae consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12; A primer pair for detection of Kingella oralis consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14; A pair of primers for detection of Porphyromonas endodontalis consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16; A pair of primers for detection of Porphyromonas gingivalis consisting of a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18; Prevotella intermedia detection primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20; Prevotella melaniogenica detection primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22; Rothia dentocariosa detection primer pair consisting of a forward primer of SEQ ID NO: 23 and a reverse primer of SEQ ID NO: 24; A pair of primers for detection of Selenomonas noxia consisting of a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26; Streptococcus salivarius detection primer pair consisting of a forward primer of SEQ ID NO: 27 and a reverse primer of SEQ ID NO: 28; Streptococcus sanguinis detection primer pair consisting of a forward primer of SEQ ID NO: 29 and a reverse primer of SEQ ID NO: 30; And a primer pair for detection of Tannerella forsythia consisting of a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32;; For detection of periodontal disease causative bacteria comprising at least one primer pair selected from the group consisting of A primer set is provided.

More specifically, the present invention may be a primer set for detection of a periodontal disease-causing bacterium comprising base sequence numbers 1 to 32 of a periodontal disease-causing bacterium represented by the following [Table 1].

[Table 1]

The primer set may include a base sequence complementary to the gene of the periodontal disease causative agent to be detected, but is not limited thereto.

The primer set is a primer set capable of amplifying all or part of the 16S rRNA gene of the periodontal disease causative bacteria.

The 16S rRNA gene is short for 16S ribosomal RNA, and refers to RNA constituting the 30S subunit of a prokaryotic ribosome.

The primers are RFLP (Restriction Fragment Length Polymorphism), RAPD (Randomly Amplified Polymorphic DNA), DAF (DNA Amplification Fingerprinting), AP-PCR (Arbitrarily Primed PCR), STS (Sequence Tagged Site), EST (Expressed Sequence Tag), SCAR Various DNAs known in the art such as (Sequence Characterized Amplified Regions), ISSR (Inter Simple Sequence Repeat), AFLP (Amplified Fragment Length Polymorphism), CAPS (Cleaved Amplified Polymorphic Sequences), PCR-SSCP (Single-Strand Conformation Polymorphism), etc. It can be designed to be suitable for analysis.

The primer set specifically amplifies a target gene of a periodontal disease-causing bacterium, and whether or not a periodontal disease-causing bacterium is detected according to the amplification or not.

The primer set is a polymerase chain reaction (PCR), a real-time polymerase chain reaction (real-time PCR), and a multiplex polymerase chain reaction (Multiplex Polymerase Chain Reaction, Multiplex PCR). It can be applied to one or more reactions selected from the group consisting of.

The periodontal disease (periodontal disease) refers to a disease of the bone or adjacent tissues of the gums. The periodontal disease begins with a bacterial infection, and when the inflammatory reaction proceeds by bacterial toxins, a periodontal pocket is formed between the gum and the teeth, and the deeper the disease, the deeper the periodontal pocket, so that the inflammation spreads to the periodontal ligament. Bone loss can occur. In addition, when the balance between bone formation and bone resorption is broken in the alveolar bone and bone resorption exceeds bone formation and bone mass decreases below the limit, alveolar bone dysplasia, a type of periodontal disease, occurs.

The periodontal disease may be gingivitis, periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy.

The alveolar bone dysplasia may be alveolar bone osteoporosis, alveolarbone osteomalacia, or alveolar bone osteopenia.

Composition for detecting periodontal disease causative bacteria

The present invention provides a composition for detecting periodontal disease causative bacteria comprising a primer set consisting of the above SEQ ID NOs: 1 to 32.

The primer set of the present invention is applied equally unless contradictory to each other in all matters mentioned in the primer set for the detection of the aforementioned periodontal disease causative bacteria.

The detection melting composition may be used for one or more reactions selected from the group consisting of polymerase chain reaction, real-time polymerase chain reaction, and multiple polymerase chain reaction.

The detection composition is a component that can be used in one or more reactions selected from the group consisting of polymerase chain reaction, real-time polymerase chain reaction, and multiple polymerase chain reaction in addition to each primer set mentioned above, such as a reaction buffer It may include one or more selected from the group consisting of dNTP, Mg ²⁺ ion, and DNA polymerase, but is not limited thereto.

The reaction buffer solution may be 1 to 10 mM TrisHCl, 10 to 40 mM KCl (pH 9.0), and the dNTP may be one or more selected from the group consisting of dATP, dTTP, dGTP, and dCTP, but limited thereto. It is not.

The detection composition may include a stabilizer and/or a non-reactive dye for experimental convenience, stabilization, and reactivity improvement.

The stabilizing agent may be a stabilizing material for more stabilizing the detection composition, and may be polyhydric alcohols, preferably 1 selected from the group consisting of glucose, glycerol, mannitol, galaxitol, glucitol and sorbitol. It can be more than a species. The polyhydric alcohols may be included in an amount of 10 to 500mM, and it is preferable to be included in an amount of 50 to 300mM. If the polyhydric alcohol exceeds 500mM, it is difficult to dissolve in an aqueous solution due to the solubility of the water-soluble polymer itself, it is difficult to achieve sufficient mixing due to the high viscosity, the volume becomes larger than necessary, and sterilization for polymerase chain reaction Dissolution is not easy when dissolved with distilled water. In addition, there is a problem in that a water-soluble polymer having a high concentration may act as a reaction inhibitor during the polymerase chain reaction. And, if it is less than 10mM, it is difficult to expect an efficient enzyme stabilization effect because the target enzyme and the surface water molecule cannot be sufficiently coated and protected.The viscosity of the solution is too low to spread widely over the entire bottom of the tube and not dry in an ideal form. There are problems and problems that the enzyme is not sufficiently protected.

In addition to polyhydric alcohols, the stabilizer may additionally be one or more selected from the group consisting of gelatin, small blue albumin, Thesit and PEG-8000 as stabilizers.

The non-reactive dye material should be selected from materials that do not affect the polymerase chain reaction, and is intended to be used for analysis or identification using a polymerase chain reaction product. Materials that satisfy these conditions may be used as water-soluble dyes such as rhodamine, tamra, lactose, bromophenol blue, xylene cyanol, bromocresol red, and cresol red. The non-reactive dye material may be included in an amount of 0.0001 to 0.01% by weight based on the total composition, and is preferably included in an amount of 0.001 to 0.005% by weight. If the non-reactive dye material is added in an amount of less than 0.0001% by weight, the concentration of the dye is low during electrophoresis after the polymerase chain reaction, making it difficult to observe the movement of the sample with the naked eye. When added as a polymerase chain reaction, there is a problem in that a high concentration water-soluble dye may act as a reaction inhibitor. In addition, there is a problem that may hinder the movement of the sample during electrophoresis.

The composition for detection may be provided in a liquid form, and is preferably in a dried state in order to increase stability, convenience of storage, and long-term storage. The drying may be performed by a known drying method such as general room temperature drying, warm drying, freeze drying, and vacuum drying, and any drying method may be used as long as the components of the detection composition are not lost. The drying method as described above may be applied differently depending on the type and amount of the enzyme used.

The detection composition is mixed in one reaction tube and then frozen or dried to be prepared and used in a stabilized state, so that a separate mixing process is not required during the polymerase chain reaction, so that errors due to mixing during the reaction can be prevented. , Stability, reactivity and storage properties can be improved.

Kit for detecting periodontal disease causative bacteria

The present invention provides a kit for detecting periodontal disease-causing bacteria comprising a primer set for detecting periodontal disease-causing bacteria.

The primer set of the present invention is applied equally unless contradictory to each other in all matters mentioned in the primer set for the detection of the aforementioned periodontal disease causative bacteria.

The detection kit may be a polymerase chain reaction, a real-time polymerase chain reaction, or a multiple polymerase chain reaction kit.

Method for detecting periodontal disease causative bacteria

The present invention provides a method for detecting periodontal disease causative bacteria comprising the following steps.

(S1) separating DNA from the biological sample;

(S2) amplifying a target sequence by treating the isolated DNA with the primer set; And

(S3) identifying the amplification product.

The primer set of the present invention is applied equally unless contradictory to each other in all matters mentioned in the primer set for the detection of the aforementioned periodontal disease causative bacteria.

The step (S1) is a step of separating DNA from a biological sample; as, a method commonly used when extracting DNA from a biological sample, for example, may be performed using a commercially available DNA extraction kit.

The step (S2) is a step of amplifying a target sequence by treating the isolated DNA with the primer set; wherein the step (S2) includes a polymerase chain reaction, a ligase chain reaction (LCR), and a Gap. -LCR, repair chain reaction, transcription-mediated amplification (TMA), self sustained sequence replication, selective amplification of target polynucleotide sequence polynucleotide sequences), consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), nucleic acid sequence-based amplification (nucleic acid) sequence based amplification (NASBA), strand displacement amplification, loop mediated isothermal amplification (LAMP), multiplex polymerase chain reaction (multiplex PCR), double polymerase chain Reaction (nested polymerase chain reaction; nested-PCR), single tube nested polymerase chain reaction (single tube nested-PCR), reverse transperase polymerase chain reaction (RT-PCR) ), inverse polymerase chain reaction chain reaction; Inverse PCR), realtime polymerase chain reaction (RT-PCR), and real-time quantitative polymerase chain reaction (RQ-PCR). The target sequence may be amplified by using, and preferably, the target sequence may be amplified using one or more methods selected from the group consisting of polymerase chain reaction, real-time polymerase chain reaction, and multiple polymerase chain reaction.

The step (S3) is a step of identifying the amplification product; As, a preliminary DNA denaturation step; Binding the selected primer set to a complementary DNA sequence; And it may be performed through the step of stretching; but is not limited thereto.

The amplified target sequence may be labeled with a detectable label. The labeling material may be a material that emits fluorescence, phosphorescence, or radioactivity, but is not limited thereto. For example, the labeling material is Cy-5, Cy-3, biotin-binding material, EDANS (5-(2'-aminoethyl) amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), tetramethylrhodamine Isocyanate (TMRITC), x-rhodamine, Texas red, SYBR green I (SYBR green I), VIC, fluorescein (FAM), carboxytetramethylrhodamine (TAMRA), carboxy-2',4,4',5',7 (HEX) ,7'-hexachloroflurescein) or BHQ3, but is not limited thereto. When amplifying the target sequence, if PCR is performed by labeling the labeling material at the 5'-end of the primer, the target sequence may be labeled with a detectable fluorescent labeling material. In addition, for labeling using a radioactive material, when a radioactive isotope such as 32P or 35S is added to the PCR reaction solution when performing PCR, the amplification product is synthesized and radioactivity is incorporated into the amplification product, so that the amplification product can be radiolabeled.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in a variety of different forms, and only the embodiments make the disclosure of the present invention complete, and common knowledge in the technical field to which the present invention pertains. It is provided to inform a person having a complete scope of the invention, and the invention is only defined by the scope of the claims.

Example 1. Preparation of primer

In order to devise a method that can specifically detect the 16 species described in the following [Table 1], the present inventors have developed a next-generation genome sequence decoding technology (Next-Generation) in order to secure the specific nucleotide sequences of the 16 species. Sequencing, NGS) was used to perform genome sequencing of the 16 representative strains to obtain a draft genome sequence, and an open reading frame from the obtained draft genome sequence. , ORF) nucleotide sequence was predicted, and the obtained ORF nucleotide sequence was compared with the nucleotide sequence of a gene known from NCBI GenBank to obtain an ORF specific to the 16 species. Subsequently, PCR primers capable of specific binding to this nucleotide sequence were prepared as shown in Table 1 below.

[Table 1]

Experimental Example 1. Confirmation of the specificity of the primer

In order to confirm the specificity of the 23 species, the primer set prepared in Example 1 was 95°C for 10 minutes, 95°C for 30 seconds, 40 cycles and 60°C for 30 seconds. A real time PCR experiment was performed. In addition, for the real time PCR, a real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, MA, Waltham, MA, USA) was used, and the results are shown in FIGS. 1 to 4.

1 to 4, it can be seen that in the case of the primer prepared in Example 1, the result (CT value) is proportional to the DNA concentration of the strain, which means that the primer specifically detects the strain. It can be said to mean that you can do it.

From the above description, it will be understood that those skilled in the art belonging to the present invention can be implemented in other specific forms without changing the technical spirit or essential features of the present invention. In this regard, the embodiments described above are illustrative in all respects, and should be understood as non-limiting.

<110> PUSAN NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> PRIMER SET FOR DETECTION OF PERIODONTAL DISEASE ASSOCIATED BACTERIA AND USING THEREOF <130> ZP20200048 <160> 32 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Campylobacter rectus_forward primer <400> 1 aatccgtaga gatcaagaag a 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Campylobacter rectus_reverse primer <400> 2 actagcgaag caacaacta 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Capnocytophaga sputigena_forward primer <400> 3 ggaactgaca gaggtagag 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Capnocytophaga sputigena_reverse primer <400> 4 acttgactcg ttagcaacta 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Eikenella corrodens_forward primer <400> 5 acctttcaga gacggaag 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Eikenella corrodens_reverse primer <400> 6 acctttcaga gacggaag 18 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium nucleatum_forward primer <400> 7 agagtctgat ccagcaatt 19 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium nucleatum_reverse primer <400> 8 gataacgctc gtgacatac 19 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium periodonticum_forward primer <400> 9 ggacagatac tgacgctaa 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fusobacterium periodonticum_reverse primer <400> 10 cggattactt atcgcgttag 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Haemophilus parainfluenzae_forward primer <400> 11 aaccttacct actcttgaca t 21 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Haemophilus parainfluenzae_reverse primer <400> 12 aacccaacat ttcacaaca 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Kingella oralis_forward primer <400> 13 aataccatta tctgctgacg 20 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Kingella oralis_reverse primer <400> 14 atttcacatc ttgcttaata aacc 24 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Porphyromonas endodontalis_forward primer <400> 15 atgatggcag atgagagtt 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Porphyromonas endodontalis_reverse primer <400> 16 atagagtcct cagcataacc 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Porphyromonas gingivalis_forward primer <400> 17 cgtaggttgt tcggtaagt 19 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Porphyromonas gingivalis_reverse primer <400> 18 agtgtcagtc gcagtatg 18 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Prevotella intermedia_forward primer <400> 19 gacgcaggat acagagat 18 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Prevotella intermedia_reverse primer <400> 20 gatggcaact aaggaaagg 19 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Prevotella melaniogenica_forward primer <400> 21 aaggcagact aataccgcat a 21 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Prevotella melaniogenica_reverse primer <400> 22 ttggctggtt cagacttc 18 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Rothia dentocariosa_forward primer <400> 23 aaggcttgac atatactgga ct 22 <210> 24 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Rothia dentocariosa_reverse primer <400> 24 tatgagtccc caccatcac 19 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Selenomonas noxia_forward primer <400> 25 aatcattggg cgtaaagg 18 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Selenomonas noxia_reverse primer <400> 26 cctctcctgt actcaagac 19 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus salivarius_forward primer <400> 27 aagtagaacg ctgaagaga 19 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus salivarius_reverse primer <400> 28 catccattgt tatgcggtat 20 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus sanguinis_forward primer <400> 29 gctaataccg cataaaattg att 23 <210> 30 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus sanguinis_reverse primer <400> 30 aggtccatct ggtagtga 18 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tannerella forsythia_forward primer <400> 31 aatccgtaga gatcaagaag a 21 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tannerella forsythia_reverse primer <400> 32 tcagtgtcag ttatacctta gt 22

Claims (9)

A primer pair for detection of Campylobacter rectus consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;
A pair of primers for detecting capnocytophaga sputigena consisting of a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;
A pair of primers for detection of Eikenella corrodens consisting of a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6;
A pair of primers for detection of Fusobacterium nucleatum consisting of a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8;
A pair of primers for detection of Fusobacterium periodonticum consisting of a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;
A pair of primers for detection of Haemophilus parainfluenzae consisting of a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
A primer pair for detection of Kingella oralis consisting of a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14;
A pair of primers for detection of Porphyromonas endodontalis consisting of a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16;
A pair of primers for detection of Porphyromonas gingivalis consisting of a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18;
Prevotella intermedia detection primer pair consisting of a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20;
Prevotella melaniogenica detection primer pair consisting of a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22;
Rothia dentocariosa detection primer pair consisting of a forward primer of SEQ ID NO: 23 and a reverse primer of SEQ ID NO: 24;
A pair of primers for detection of Selenomonas noxia consisting of a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26;
Streptococcus salivarius detection primer pair consisting of a forward primer of SEQ ID NO: 27 and a reverse primer of SEQ ID NO: 28;
Streptococcus sanguinis detection primer pair consisting of the forward primer of SEQ ID NO: 29 and the reverse primer of SEQ ID NO: 30; And
A primer set for detection of periodontal disease causative bacteria comprising; a pair of primers for detection of Tannerella forsythia consisting of a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32. The method of claim 1,
The periodontal disease is gingivitis, periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy. The method of claim 2,
The alveolar bone dysplasia is a set of primers for detecting the cause of periodontal disease, which is alveolar bone osteomalacia, alveolarbone osteomalacia, or alveolar bone osteopenia. A composition for detecting periodontal disease causative bacteria comprising the primer set according to any one of claims 1 to 3. The method of claim 4,
The composition consists of a polymerase chain reaction (PCR), a real-time polymerase chain reaction (real-time PCR), and a multiplex polymerase chain reaction (Multiplex Polymerase Chain Reaction, Multiplex PCR). A composition for detecting periodontal disease causative bacteria used in one or more reactions selected from the group. A kit for detecting periodontal disease causative bacteria comprising the primer set according to any one of claims 1 to 3. The method of claim 6,
The kit is a kit for polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR) or multiplex polymerase chain reaction (Multiplex Polymerase Chain Reaction, Multiplex PCR). Kit for detection of bacteria that cause periodontal disease (S1) separating DNA from the biological sample;
(S2) amplifying a target sequence by treating the isolated DNA with a primer set according to any one of claims 1 to 3; And
(S3) the step of identifying the amplification product; a method for detecting periodontal disease causative bacteria comprising. The method of claim 8,
The detection method includes polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), and multiplex polymerase chain reaction (multiplex polymerase chain reaction, multiplex PCR). A method of detecting one or more periodontal disease causative bacteria selected from the group consisting of.

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