KR102573767B1 - Information provision method and kit for diagnosis of periodontal disease in Korean - Google Patents

Abstract

The present invention relates to a method and kit for providing information for diagnosing periodontal disease in Koreans, and specifically, a microorganism of the genus Actinomyces in a sample derived from the oral cavity of a subject; Fretibacterium genus microorganisms; Prevotella dentalis ( Prevotella dentalis ); Tannerella Forsythia ( Tannerella forsythia ); Eubacterium nodatum ; Mosquitobacterium ( Mogibacterium ) Genus microorganisms; Porphyromonas endodontalis ( Porphyromonas endodontalis ); Treponema genus microorganisms; Filifactor alocis ; Treponema denticola ; Treponema socranskii ; Eubacterium saphenum ; Haemophilus parainfluenzae ; Oribacterium ( Oribacterium ) genus microorganisms; Porphyromonas gingivalis ( Porphyromonas gingivalis ); And a method for providing information for diagnosing periodontal disease in Koreans for detecting microorganisms of the genus Mycoplasma , and a primer for detecting the 16 microorganisms - relates to a kit for diagnosing periodontal disease in Koreans, including a probe set.
The information provision method and kit of the present invention fully consider the comprehensive oral microbial environment of Koreans, and when used, periodontal disease of Koreans can be accurately and efficiently diagnosed.

Description

Information provision method and kit for diagnosis of periodontal disease in Korean}

The present invention relates to a method and kit for providing information for diagnosing periodontal disease in Koreans, and specifically, a microorganism of the genus Actinomyces in a sample derived from the oral cavity of a subject; Fretibacterium genus microorganisms; Prevotella dentalis ( Prevotella dentalis ); Tannerella Forsythia ( Tannerella forsythia ); Eubacterium nodatum ; Mosquitobacterium ( Mogibacterium ) Genus microorganisms; Porphyromonas endodontalis ( Porphyromonas endodontalis ); Treponema genus microorganisms; Filifactor alocis ; Treponema denticola ; Treponema socranskii ; Eubacterium saphenum ; Haemophilus parainfluenzae ; Oribacterium ( Oribacterium ) genus microorganisms; Porphyromonas gingivalis ( Porphyromonas gingivalis ); And a method for providing information for diagnosing periodontal disease in Koreans for detecting microorganisms of the genus Mycoplasma , and a primer for detecting the 16 microorganisms - relates to a kit for diagnosing periodontal disease in Koreans, including a probe set.

Periodontal disease is a chronic inflammatory disease in which periodontal tissue is destroyed by bacteria that cause periodontal disease in the oral cavity and toxins produced by these bacteria. include It is known that the direct cause of periodontal disease is bacterial plaque, tartar, smoking, etc., and is indirectly related to lifestyle and general health as well as socioeconomic level.

More than 700 species of microorganisms form a colony in the human oral cavity, competing with each other to achieve homeostasis. Periodontal disease is a chronic disease. Chronic diseases are difficult to cure once they develop and require lifelong management, and in many cases, biological, environmental, and socioeconomic factors act in a complex way. In addition, holistic management for treatment is required because diseases are influenced by common action factors. Periodontal disease is a 'silent disease' that has no symptoms in the early stages and is easy to overlook even if there are minor symptoms. As age increases, the incidence increases, and according to the statistics of frequent diseases in 2019, gingivitis and periodontal disease ranked first, with a 6.7% increase in patients compared to last year.

Several studies have reported that there are ethnic differences in the incidence and progression of periodontal disease, and that this is due to differences in the causative bacteria of periodontitis and differences in the degree of host response to specific periodontal disease causative bacteria according to ethnic characteristics of periodontitis. Therefore, analyzing the distribution of periodontal disease causative bacteria in a specific country or ethnic group can help predict the frequency of periodontal disease and establish an optimal treatment plan.

Currently, for the diagnosis of periodontal disease in Korea, real-time polymerase chain reaction (Real-time Polymerase Chain Reaction , Real-time PCR ) are being used for qualitative and quantitative analysis, but most of these services or related products do not consider the relevance according to countries and ethnicities, and the comprehensive oral microbial environment is not considered.

Accordingly, the present inventors have attempted to develop a method and related products that can more accurately and efficiently diagnose or use periodontal disease in Koreans by sufficiently considering the comprehensive oral microbial environment of Koreans.

Seonmi Kim, et al. "Quantitative analysis of periodontal disease causative bacteria using real-time PCR." Korean Journal of Pediatric Dentistry 35.3 (2008): 494-503. Kyung Hee Medicine: Vol. 32 Vol. 1, J Kyung Hee Univ Med Cent: Vol. 32, no. 1, 2017. Yun, Jeong-Ho, et al, 2008, The Korean Academy Of Periodontology, 143-152. J Clin Periodontol. 2000 Sep;27(9):648-57. Pereira JV, Leomil L, Rodrigues-Albuquerque F, Pereira JO, Brazilian dental journal. 2012;23(4):409-16. Van Hoogmoed CG, Geertsema-Doornbusch GI, Teughels W, Quirynen M, Busscher HJ, Van der Mei HC. Oral Microbiol Immun. 2008;23(1):43-8. Khemwong T, Kobayashi H, Ikeda Y, Matsuura T, Sudo T, Kano C, et al. Plos one. 2019;14(6). Casarin RC, Saito D, Santos VR, Pimentel SP, Duarte PM, Casati MZ, et al. Brazilian journal of microbiology. 2012;43(3):931-7. Zhou X, Li Y. Atlas of oral microbiology: From healthy microflora to disease: Academic press; 2015. Kwek, H. S. N., M. Wilson, and H. N. Newman Journal of clinical periodontology 17.2 (1990): 119-122. Hiranmayi KV, Sirisha K, Ramoji Rao MV, Sudhakar P. Journal of pharmacy & bioallied sciences. 2017;9(3):155-63. Korean Journal of Periodontal Science 2008;38:543-550. Lee M-Y, Kwon E-Y, Kim H-J, Lee J-Y, Joo J-Y. Journal of Dental Rehabilitation and Applied Science. 2018;34(3):186-95. J Periodontol. 2001 Oct; 72(10):1354-63. Marchesan J, Jiao Y, Schaff RA, Hao J, Morelli T, Kinney JS, et al. Molecular oral microbiology. 2016;31(3):243-58. Jia, Lu, et al. (2019) Frontiers in cellular and infection microbiology 9.

The main object of the present invention is to provide an information providing method and kit that can be used to more accurately and efficiently diagnose periodontal disease in Koreans by fully considering the comprehensive oral microbial environment of Koreans.

According to one aspect of the present invention, the present invention is a primer of SEQ ID NO: 1, a primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 35 in a sample derived from the oral cavity of the subject, detected by PCR using Actinomyces ( Actinomyces ) microorganisms ; A microorganism of the genus Fretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; Prevotella dentalis ( Prevotella dentalis ); Tannerella Forsythia ( Tannerella forsythia ); Eubacterium nodatum ; A microorganism of the genus Mogibacterium detected by PCR using the primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; Porphyromonas endodontalis ( Porphyromonas endodontalis ); A microorganism of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; Filifactor alocis ; Treponema denticola ; Treponema socranskii ; Eubacterium saphenum ; Haemophilus parainfluenzae ; A microorganism of the genus Oribacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; Porphyromonas gingivalis ( Porphyromonas gingivalis ); And a primer of SEQ ID NO: 31, a primer of SEQ ID NO: 32, and a microorganism of the genus Mycoplasma detected by PCR using a probe of SEQ ID NO: 50; provides a method for providing information for diagnosing periodontal disease in Koreans.

In the information providing method of the present invention, the primers of SEQ ID NO: 1, the sequences of SEQ ID NO: 1 for the detection of microorganisms of the genus Actinomyces detected by PCR using the primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the probe of SEQ ID NO: 35 PCR using the primer of SEQ ID NO: 2 and the probe of SEQ ID NO: 35; The primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the primers of SEQ ID NO: 4 and SEQ ID NO: 36 for detection of microorganisms of the genus Pretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36 PCR using probes; PCR using the primers of SEQ ID NO: 5, the primers of SEQ ID NO: 6, and the probe of SEQ ID NO: 37 for detection of the Prevotella dentalis; PCR using the primers of SEQ ID NO: 7, the primers of SEQ ID NO: 8, and the probe of SEQ ID NO: 38 for the detection of the Tanerella forsythia; PCR using the primers of SEQ ID NO: 9, the primers of SEQ ID NO: 10, and the probe of SEQ ID NO: 39 for detection of the Eubacterium nodatum; The primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40 are used to detect microorganisms of the genus Mosquitobacterium detected by PCR using the primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40 PCR using; PCR using the primers of SEQ ID NO: 13, the primers of SEQ ID NO: 14, and the probe of SEQ ID NO: 41 for detection of the Porphyromonas endodonthalis; Primers of SEQ ID NO: 15, primers of SEQ ID NO: 16, and probes of SEQ ID NO: 42 for detection of microorganisms of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42 PCR using; PCR using the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43 for detection of the Philifactor allosis; PCR using the primers of SEQ ID NO: 19, the primers of SEQ ID NO: 20, and the probe of SEQ ID NO: 44 for detection of the Treponema denticola; PCR using the primers of SEQ ID NO: 21, the primers of SEQ ID NO: 22, and the probe of SEQ ID NO: 45 for detection of the Treponema sokranskii; PCR using the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46 for detection of the Eubacterium sapenum; PCR using the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47 for detection of the Haemophilus parainfluenza; The primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probes of SEQ ID NO: 28 and the probes of SEQ ID NO: 28 for detection of microorganisms of the genus Orybacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48 PCR using; PCR using the primers of SEQ ID NO: 29, the primers of SEQ ID NO: 30, and the probe of SEQ ID NO: 49 for detection of the Porphyromonas gingivalis; The primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50 were used to detect the microorganisms of the genus Mycoplasma detected by PCR using the primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50. It is preferable to perform PCR;

In the information providing method of the present invention, PCR for detecting microorganisms of the genus Actinomyces detected by PCR using the primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the probe of SEQ ID NO: 35; PCR for detecting microorganisms of the genus Pretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; PCR for detection of the Prevotella dentalis; and PCR for detecting the Tanerella fossitia; performed in one reaction solution, and PCR for detecting the Eubacterium nodatum; PCR for detecting microorganisms of the genus Mosquitobacterium detected by PCR using the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; PCR for detection of the Porphyromonas endodonthalis; and PCR for detecting microorganisms of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; carried out in one reaction solution, and the Philifactor allo PCR for detection of cis; PCR for detection of the Treponema denticola; and PCR for detection of Treponema sokranskii; performed in one reaction solution, and PCR for detection of Eubacterium sapenum; PCR for detection of the Haemophilus parainfluenza; PCR for detecting microorganisms of the genus Oribacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; And PCR for the detection of Porphyromonas gingivalis; is preferably carried out in one reaction solution.

In the information providing method of the present invention, PCR using the primers of SEQ ID NO: 33, the primers of SEQ ID NO: 34, and the probe of SEQ ID NO: 51 for detection of Gram-positive microorganisms; And PCR using the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, and the probe of SEQ ID NO: 52 for detection of Gram-negative microorganisms; PCR for detecting microorganisms of the genus Mycoplasma detected by PCR using a probe of; PCR for detection of the Gram-positive microorganism; And PCR for detection of the gram-negative microorganisms; it is preferable to perform in one reaction solution.

According to another aspect of the present invention, a first primer-probe set including a primer of SEQ ID NO: 1, a primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 35; a second primer-probe set comprising the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; a third primer-probe set comprising the primers of SEQ ID NO: 5, the primers of SEQ ID NO: 6, and the probe of SEQ ID NO: 37; a fourth primer-probe set comprising the primers of SEQ ID NO: 7, the primers of SEQ ID NO: 8, and the probe of SEQ ID NO: 38; a fifth primer-probe set including the primer of SEQ ID NO: 9, the primer of SEQ ID NO: 10, and the probe of SEQ ID NO: 39; a sixth primer-probe set comprising the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; a seventh primer-probe set comprising the primers of SEQ ID NO: 13, the primers of SEQ ID NO: 14, and the probe of SEQ ID NO: 41; an eighth primer-probe set comprising the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; a ninth primer-probe set comprising the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43; a 10th primer-probe set comprising the primer of SEQ ID NO: 19, the primer of SEQ ID NO: 20, and the probe of SEQ ID NO: 44; an 11th primer-probe set comprising the primer of SEQ ID NO: 21, the primer of SEQ ID NO: 22, and the probe of SEQ ID NO: 45; a twelfth primer-probe set comprising the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46; a thirteenth primer-probe set comprising the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47; a 14th primer-probe set comprising the primer of SEQ ID NO: 27, the primer of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; a 15th primer-probe set comprising the primer of SEQ ID NO: 29, the primer of SEQ ID NO: 30, and the probe of SEQ ID NO: 49; and a 16th primer-probe set comprising the primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50; a PCR kit for diagnosing periodontal disease in Koreans is provided.

In the kit of the present invention, the first primer-probe set, the second primer-probe set, the third primer-probe set, and the fourth primer-probe set are included in one composition, and the fifth primer-probe set , The 6th primer-probe set, the 7th primer-probe set and the 8th primer-probe set are included in one composition, the 9th primer-probe set, the 10th primer-probe set and the 11th primer-probe set in one composition, and the twelfth primer-probe set, the 13th primer-probe set, the 14th primer-probe set, and the 15th primer-probe set are preferably included in one composition.

The kit of the present invention further comprises a 17th primer-probe set including the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, the probe of SEQ ID NO: 51, and the probe of SEQ ID NO: 52, wherein the 16th primer- It is preferable to include the probe set and the 17th primer-probe set in one composition.

The information provision method and kit of the present invention fully consider the comprehensive oral microbial environment of Koreans, and when used, periodontal disease of Koreans can be accurately and efficiently diagnosed.

Figure 1 shows the results of performing a single real-time PCR with each of the primers and probes of the present invention using all 16 plasmid DNAs (excluding total bacteria) for each target microorganism as a template. M: Size Marker, Pd: Prevotella Dentalis, Tf: Tanerella Forsythia, En: Eubacterium Nodatum, Pe: Porphyromonas Endodonthalis, Fa: Philifactor Allosis, Td: Treponema Denti Cola, Ts: Treponema sokranskii, Es: Eubacterium sapenum, Hp: Haemophilus parainfluenza, Pg: Porphyromonas gingivalis.
2 to 17 are results of single real-time PCR using each primer-probe of the present invention and plasmid DNA for the corresponding target microorganism. In order from FIGS. 2 to 17, Porphyromonas Endodonthalis, Treponema sokranskii, Prevotella dentalis, Eubacterium sapenum, Treponema denticola, Eubacterium nodatium, Tanerella posi Tia, Philifactor alosis, Porphyromonas gingivalis, microorganisms of the genus Treponema, microorganisms of the genus Mycoplasma, microorganisms of the genus Pretibacterium, microorganisms of the genus Mosquitobacterium, Haemophilus parainfluenza, Actinomyces These are the results for microorganisms in the genus Orybacterium genus.
18 to 22 are the results of performing multiplex real-time PCR with each set of Table 4 using each primer-probe of the present invention and plasmid DNA for the corresponding target microorganism. Pd: Prevotella dentalis, Tf: Tanerella fosythia, En: Eubacterium nodatium, Pe: Porphyromonas endodontalis, Fa: Philifactor alosis, Td: Treponema denticola, Ts: Tre Ponema Sokranskii, Es: Eubacterium sapenum, Hp: Haemophilus parainfluenza, Pg: Porphyromonas gingivalis.

The method for providing information for diagnosing periodontal disease in Koreans of the present invention is a method for detecting Actinomyces ( Actinomyces ) genus microorganism; A microorganism of the genus Fretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; Prevotella dentalis ( Prevotella dentalis ); Tannerella Forsythia ( Tannerella forsythia ); Eubacterium nodatum ; A microorganism of the genus Mogibacterium detected by PCR using the primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; Porphyromonas endodontalis ( Porphyromonas endodontalis ); A microorganism of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; Filifactor alocis ; Treponema denticola ; Treponema socranskii ; Eubacterium saphenum ; Haemophilus parainfluenzae ; A microorganism of the genus Oribacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; Porphyromonas gingivalis ( Porphyromonas gingivalis ); And the primers of SEQ ID NO: 31, the primers of SEQ ID NO: 32, and the probe of SEQ ID NO: 50 detected by PCR using Mycoplasma ( Mycoplasma ) genus microorganisms; characterized by detecting.

The microorganisms were selected through a study on the oral microbial environment targeting Koreans, specifically, a study on the oral microbial environment targeting Koreans with a healthy oral environment without periodontal disease and Koreans with periodontal disease. Microorganisms of the genus Pretibacterium, Prevotella dentalis, Tanerella fossitia, Eubacterium nodatium, Microorganisms of the genus Mosquitobacterium, Porphyromonas endodontalis, Microorganisms of the genus Treponema, Philifactor allosis, Trepo Microorganisms of the genus Nema denticola, Treponema sokranskyi, Eubacterium sapenum, Porphyromonas gingivalis, and Mycoplasma are selected as harmful bacteria that cause periodontal disease, and the microorganisms of the genus Actinomyces, duck Microorganisms of the genus Bacterium and Haemophilus parainfluenza were selected as common flora present in healthy subjects.

If the balance of microorganisms in the oral cavity is broken, such as a large number of microorganisms selected as harmful bacteria and a small number of microorganisms selected as the normal flora detected in oral samples of the subject, that is, a Korean subject, the subject is classified as having a high risk of periodontal disease Conversely, if the balance of microorganisms in the oral cavity is maintained, such as a small number of microorganisms selected as harmful bacteria and a large number of microorganisms selected as normal bacteria in the sample derived from the oral cavity of the subject, the subject's risk of periodontal disease can be classified as low. there is. For example, the risk of periodontal disease may be indicated by assigning '+1 point' to each detected harmful bacteria and assigning '-1 point' to each detected common bacteria.

In the present invention, the sample derived from the oral cavity of the subject refers to a biological sample derived from the oral cavity of the subject, and is preferably the subject's saliva (saliva) or gargle liquid.

In the present invention, the microorganisms specified as being detected by PCR using specific primers and probes refer to microorganisms whose genomes contain sequences detected by PCR using the corresponding primers and probes, for example, real-time PCR.

In the present invention, the microorganism of the genus Actinomyces may be a microorganism whose genome contains a sequence registered as accession number KF933792.1 in the GenBank database, Actinomyces sp. It may be ChDC B642.

In the present invention, the microorganism of the genus Pretibacterium may be a microorganism whose genome contains a sequence registered as registration number KT895067.1 in the GenBank database.

In the present invention, the Prevotella dentalis may be a microorganism whose genome has a sequence registered as registration number NR_102481.1 in the GenBank database, and may be Prevotella dentalis DSM 3688.

In the present invention, the Tannerella forsythia may be a microorganism whose genome has a sequence registered as registration number NR_074140.1 in the GenBank database, and may be Tannerella forsythia strain ATCC 43037.

In the present invention, the Eubacterium nodatum may be a microorganism whose genome contains a sequence registered as registration number GU424720.1 in the GenBank database.

In the present invention, the microorganism of the genus Mogibacterium may be a microorganism whose genome contains a sequence registered as registration number MT482651.1 in the GenBank database, Mogibacterium sp. strain KCOM 3331.

In the present invention, the Porphyromonas endodonthalis may be a microorganism whose genome contains a sequence registered as registration number LT676548.1 in the GenBank database.

In the present invention, the microorganism of the genus Treponema may be a microorganism whose genome has a sequence registered as registration number GU408605.1 in the GenBank database, and Treponema sp. It may be oral taxon 230.

In the present invention, the Filifactor alocis may be a microorganism whose genome contains a sequence registered as registration number MT322993.1 in the GenBank database, and may be Filifactor alocis strain KCOM 3147 (=JS254).

In the present invention, the Treponema denticola may be a microorganism whose genome contains a sequence registered as registration number NR_036899.1 in the GenBank database.

In the present invention, the Treponema sokranskii may be a microorganism whose genome contains a sequence registered as registration number LT698215.1 in the GenBank database.

In the present invention, the Eubacterium saphenum may be a microorganism whose genome has a sequence registered as registration number NR_026031.1 in the GenBank database, and may be Eubacterium saphenum strain 164-47.

In the present invention, the Haemophilus parainfluenza may be a microorganism whose genome has a sequence registered as registration number MT096513.1 in the GenBank database, and may be Haemophilus parainfluenzae strain Ch6.

In the present invention, the microorganism of the genus Oribacterium may be a microorganism whose genome contains a sequence registered as registration number LT698418.1 in the GenBank database.

In the present invention, the Porphyromonas gingivalis may be a microorganism whose genome has a sequence registered as registration number KR074427.1 in the GenBank database, and may be Porphyromonas gingivalis strain CP41.

In the present invention, the microorganism of the genus Mycoplasma may be a microorganism whose genome contains a sequence registered as registration number AM420094.1 in the GenBank database.

In the present invention, PCR is preferably performed using a primer of SEQ ID NO: 1, a primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 35 to detect microorganisms of the genus Actinomyces, and detection of microorganisms of the genus Pretibacterium For this, PCR is preferably performed using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36, and the primers of SEQ ID NO: 5, SEQ ID NO: PCR was performed using the primer of SEQ ID NO: 6 and the probe of SEQ ID NO: 37, and PCR was preferably performed using the primer of SEQ ID NO: 7, the primer of SEQ ID NO: 8, and the probe of SEQ ID NO: 38 to detect the Tanerella fossitia. And, for the detection of the Eubacterium nodatum, PCR is preferably performed using the primer of SEQ ID NO: 9, the primer of SEQ ID NO: 10, and the probe of SEQ ID NO: 39, and preferably for the detection of microorganisms of the genus Mosquitobacterium Preferably, PCR is performed using the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40, and preferably, the primers of SEQ ID NO: 13 and SEQ ID NO: 14 are used to detect the Porphyromonas endodonthalis. PCR was performed using primers and a probe of SEQ ID NO: 41, and PCR was preferably performed using a primer of SEQ ID NO: 15, a primer of SEQ ID NO: 16, and a probe of SEQ ID NO: 42 to detect the microorganism of the genus Treponema, , PCR is preferably performed using the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43 to detect the Philifactor allosis, and to detect the Treponema denticola, preferably PCR was performed using the primers of SEQ ID NO: 19, the primers of SEQ ID NO: 20, and the probe of SEQ ID NO: 44, and the primers of SEQ ID NO: 21, the primers of SEQ ID NO: 22 and PCR was performed using the probe of SEQ ID NO: 45, and PCR was preferably performed using the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46 for detection of the Eubacterium sapenum, For the detection of Haemophilus parainfluenza, PCR is preferably performed using the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47, and preferably for the detection of microorganisms of the genus Orybacterium. Performs PCR using the primer of SEQ ID NO: 27, the primer of SEQ ID NO: 28, and the probe of SEQ ID NO: 48, and preferably the primer of SEQ ID NO: 29 and the primer of SEQ ID NO: 30 for detection of the Porphyromonas gingivalis And PCR is performed using the probe of SEQ ID NO: 49, and PCR is preferably performed using primers of SEQ ID NO: 31, primers of SEQ ID NO: 32, and probes of SEQ ID NO: 50 to detect the microorganisms of the genus Mycoplasma.

When using the primers and probes as described above, PCR for detecting microorganisms of the genus Actinomyces; PCR for detection of microorganisms of the genus Pretibacterium; PCR for detection of the Prevotella dentalis; and PCR for detecting the Tanerella fossitia; PCR for detecting the Eubacterium nodatum; PCR for detecting microorganisms of the genus Mosquitobacterium; PCR for detection of the Porphyromonas endodonthalis; and PCR for detecting the microorganism of the genus Treponema; may be performed in one reaction solution, and the PCR for detecting the Philifactor allosis; PCR for detection of the Treponema denticola; and PCR for detection of the Treponema sokranskii; PCR for detection of the Eubacterium sapenum; PCR for detection of the Haemophilus parainfluenza; PCR for detecting microorganisms of the genus Oribacterium; And PCR for the detection of Porphyromonas gingivalis; can be carried out in one reaction solution. This is possible because the PCR primers and probes presented as capable of being performed in one reaction solution are designed to have high specificity and sensitivity without interfering with each other. Accordingly, since more rapid or easy detection is possible using the corresponding primers and probes presented above, it is possible to provide more efficient information for diagnosing periodontal disease in Koreans.

PCR using the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, and the probe of SEQ ID NO: 51 for detection of Gram-positive microorganisms in addition to the PCR for detecting each microorganism in the present invention; and PCR using the primers of SEQ ID NO: 33, the primers of SEQ ID NO: 34, and the probe of SEQ ID NO: 52 for detection of Gram-negative microorganisms. These PCRs are for the role of a control group, and if these PCRs are additionally performed, the reliability of the information can be increased.

In addition, PCR for detection of the Gram-positive microorganism; and PCR for detection of gram-negative microorganisms; can be performed in one reaction solution together with PCR for detection of microorganisms of the genus Mycoplasma.

In the present invention, PCR is a 3-step PCR that repeats 3 steps of denaturation - annealing - extension or 2-step PCR that simultaneously performs linking and extension. ) can be used. Preferably, a two-step PCR that can reduce the reaction time is used.

The PCR condition is preferably a condition in which a denaturation step at 95°C for 15 seconds and a binding and elongation step at 60°C for 30 seconds are repeated 40 times (cycles).

Preferably, a UDG (Uracil-DNA Glycosylase) reaction step is added before PCR is performed under the above conditions to prevent result errors due to contamination.

The information provision method of the present invention is effective as an information provision method for diagnosing periodontitis in particular among periodontal diseases.

The kit for diagnosing periodontal disease in Koreans of the present invention is a kit for diagnosing periodontal disease in Koreans by detecting the periodontal disease-related microorganisms by PCR, preferably real-time PCR. a first primer-probe set including probe number 35; a second primer-probe set comprising the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; a third primer-probe set comprising the primers of SEQ ID NO: 5, the primers of SEQ ID NO: 6, and the probe of SEQ ID NO: 37; a fourth primer-probe set comprising the primers of SEQ ID NO: 7, the primers of SEQ ID NO: 8, and the probe of SEQ ID NO: 38; a fifth primer-probe set including the primer of SEQ ID NO: 9, the primer of SEQ ID NO: 10, and the probe of SEQ ID NO: 39; a sixth primer-probe set comprising the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; a seventh primer-probe set comprising the primers of SEQ ID NO: 13, the primers of SEQ ID NO: 14, and the probe of SEQ ID NO: 41; an eighth primer-probe set comprising the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; a ninth primer-probe set comprising the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43; a 10th primer-probe set comprising the primer of SEQ ID NO: 19, the primer of SEQ ID NO: 20, and the probe of SEQ ID NO: 44; an 11th primer-probe set comprising the primer of SEQ ID NO: 21, the primer of SEQ ID NO: 22, and the probe of SEQ ID NO: 45; a twelfth primer-probe set comprising the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46; a thirteenth primer-probe set comprising the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47; a 14th primer-probe set comprising the primer of SEQ ID NO: 27, the primer of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; a 15th primer-probe set comprising the primer of SEQ ID NO: 29, the primer of SEQ ID NO: 30, and the probe of SEQ ID NO: 49; and a 16th primer-probe set including the primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50.

The first primer-probe set is a primer-probe set for detecting microorganisms of the genus Actinomyces, and the second primer-probe set is a primer-probe set for detecting microorganisms of the genus Pretibacterium, The third primer-probe set is a primer-probe set for detection of Prevotella dentalis, the fourth primer-probe set is a primer-probe set for detection of Tanerella Forsythia, and the fifth The primer-probe set is a primer-probe set for detecting Eubacterium nodatum, the sixth primer-probe set is a primer-probe set for detecting microorganisms of the genus Mosquitobacterium, and the seventh primer-probe set is for detecting microorganisms of the genus Mosquitobacterium. The probe set is a primer-probe set for detecting Porphyromonas endodonthalis, the eighth primer-probe set is a primer-probe set for detecting a microorganism of the genus Treponema, and the ninth primer-probe set set is a primer-probe set for detection of the Philifactor allosis, the 10th primer-probe set is a primer-probe set for detection of the Treponema denticola, and the 11th primer-probe set is the A primer-probe set for detection of Treponema sokranskii, the twelfth primer-probe set is a primer-probe set for detection of Eubacterium sapenum, and the thirteenth primer-probe set is for detection of the H. A primer-probe set for detection of Mophilus parainfluenza, the 14th primer-probe set is a primer-probe set for detection of microorganisms of the genus Orybacterium, and the 15th primer-probe set is for the foreskin A primer-probe set for detecting Lomonas gingivalis, and the 16th primer-probe set is a primer-probe set for detecting microorganisms of the genus Mycoplasma.

As described above, PCR for detection of microorganisms of the genus Actinomyces; PCR for detection of microorganisms of the genus Pretibacterium; PCR for detection of the Prevotella dentalis; and PCR for detecting the Tanerella fossitia; PCR for detecting the Eubacterium nodatum; PCR for detecting microorganisms of the genus Mosquitobacterium; PCR for detection of the Porphyromonas endodonthalis; and PCR for detecting the microorganism of the genus Treponema; may be performed in one reaction solution, and the PCR for detecting the Philifactor allosis; PCR for detection of the Treponema denticola; and PCR for detection of the Treponema sokranskii; PCR for detection of the Eubacterium sapenum; PCR for detection of the Haemophilus parainfluenza; PCR for detecting microorganisms of the genus Oribacterium; and PCR for detection of P. gingivalis; since the primers and probes are designed so that they can be performed in one reaction solution, the kit of the present invention includes the first primer-probe set, the second primer -Including a probe set, a third primer-probe set and a fourth primer-probe set in one composition, the fifth primer-probe set, the sixth primer-probe set, the seventh primer-probe set, and the eighth primer -Including a probe set in one composition, including the 9th primer-probe set, the 10th primer-probe set, and the 11th primer-probe set in one composition, the 12th primer-probe set, the 13th primer-probe set The primer-probe set, the 14th primer-probe set, and the 15th primer-probe set may be included in one composition. In this case, the composition is, for example, a composition for PCR reaction, and may further include materials necessary for PCR reaction, such as polymerase, dNTP, buffer, etc., in addition to the primer-probe set.

In addition to the above primer-probe set, the kit of the present invention further includes a 17th primer-probe set including the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, the probe of SEQ ID NO: 51, and the probe of SEQ ID NO: 52. can include This is a primer-probe set for detecting gram-positive microorganisms and gram-negative microorganisms, can serve as a control group, and can contribute to increasing the reliability of diagnosis.

As described above, PCR for detection of the gram-positive microorganism; And PCR for detection of gram-negative microorganisms; PCR for detection of microorganisms of the genus Mycoplasma; Since the primers and probes of the present invention are designed to perform in one reaction solution, the kit of the present invention The 16th primer-probe set and the 17th primer-probe set may be included in one composition. The composition at this time is also, for example, a composition for PCR reaction, and materials necessary for PCR reaction, for example, polymerase, dNTP, buffer, etc. may be further included in addition to the primer-probe set.

Primers and probes in the present invention may be labeled with a labeling material commonly used in this field. Both primers and probes may be labeled, but typically only the probes are labeled. There is no particular restriction on the labeling material used here, but the primer-probe sets presented as being able to be performed in one reaction solution are labeled with different labeling materials so that they can be detected separately from each other in one PCR reaction. it is desirable For example, the probe of the first primer-probe set (probe of SEQ ID NO: 35), the probe of the second primer-probe set (probe of SEQ ID NO: 36), the probe of the third primer-probe set (probe of SEQ ID NO: 37) ) and the probe of the fourth primer-probe set (probe of SEQ ID NO: 38) are labeled with different labeling substances. When one probe is labeled with several types of labeling substances, only some of them can be differentiated from other sets of probes.

The kit of the present invention is preferably a kit for diagnosing periodontitis in particular among periodontal diseases.

In addition to the above primer sets, the kit of the present invention may further include reagents, tools, and the like necessary for easily performing PCR. For example, polymerase, dNTP, buffer, etc. necessary for PCR may be included, and reagents, tools, etc. necessary for extracting DNA from a sample may be further included.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are intended to illustrate the present invention only, the scope of the present invention is not to be construed as being limited by these examples.

[Example]

Example 1

1.1 Recruitment of subjects and collection of oral samples (saliva)

Subject selection criteria were based on the criteria proposed by the American Academy of Periodontology (APP) in 1999. A total of 250 adults who visited the periodontal department of Pusan National University Dental Hospital for examination and treatment were diagnosed as healthy people and patients with chronic periodontitis. Patients with chronic periodontitis were divided into 4 groups and classified into early, middle, and late periodontitis according to the severity of the disease. The severity of chronic periodontitis was divided into early if 1 or 2, intermediate if 3 or 4, and late if 5 or more based on CAL (clinimcal attachment level) (Table 1). In addition, after sufficiently understanding the research for the present invention from the selected subjects, a written consent form and a clinical research survey questionnaire voluntarily agreeing to participate in the study were obtained.

Subjects who had a systemic disease that could affect their oral care hygiene ability, who had received dental treatment 6 months before the time of examination, who withdrew their consent to participate in the study, or who had less than 20 teeth were excluded. In the case of women, pregnant or lactating women were also excluded. The subject's saliva was collected by providing a gargle solution, gargling for 30 seconds, and then collecting the gargle solution in a designated container, and storing it in a refrigerator until the experiment.

Table 1 is a list by group of the present invention.

1.2 Microbial DNA extraction from oral samples (saliva)

Genomic DNA was extracted from a refrigerated oral sample (saliva) sample using the GeneAll Exgene™ Clinic SV kit (GeneAll Biotechnology, Korea) according to the manufacturer's instructions. After confirming the concentration and quality (260/280 ratio, 260/230 ratio) with a microplate reader Epoch2 (BioTek, USA), the samples were stored frozen at -20°C until used for next-generation sequencing (NGS).

Example 2

2.1 Oral microbiome analysis through next-generation sequencing (NGS)

Next-generation sequencing (NGS) was performed to identify the oral microbiome related to periodontitis with the DNA sample obtained by the method of Example 1. The diversity of the communities between each group was confirmed, and based on this data, candidates for oral microorganisms with significant differences between the healthy group and the periodontitis patient group (early, middle, and late stages) were selected by referring to the databases Greengenes and SILVA.

In terms of species, nine species showing significant results between the healthy group and the periodontitis group (early, middle, and late stages) were selected as candidates. In the Greengenes results, Porphyromonas endodontalis , Treponema socranskii , Haemophilus parainfluenzae , and Neisseria subflava were selected, In SILVA, Porphyromonas endodontalis , Prevotella dentalis , Eubacterium saphenum , Treponema denticola , Eubacterium brachy ) and Eubacterium nodatum were selected (see Table 2).

At the genus level, 11 species showing significant results between the healthy group and the periodontitis group (early, middle, and late stages) were selected as candidates. In the Greengenes results, Filifactor , Treponema , Tannerella , Mycoplasma , Porphyromonas , Prevotella , Oribacter The genus Oribacterium , the genus Mogibacterium , the genus Actinomyces , and the genus Rothia were selected, and in SILVA, the genus Filifactor , the genus Treponema , The genus Tannerella , Mycoplasma , Porphyromonas , Mogibacterium , and Fretibacterium were selected (see Table 3).

Tables 2 and 3 are lists of selected strains.

2.2 Selection of oral bacteria related to periodontitis

Oral bacteria related to periodontitis were selected by referring to the non-patent literature among the oral bacteria candidates selected by the method of 2.1 above. In the case of the genus unit, when the corresponding genus was detected or the NGS sequence was searched for as a single species, the corresponding species was selected.

The bacteria finally selected by referring to the non-patent literature are as follows. Treponema denticola (Non-Patent Document 0013), which forms harmful factors in the oral cavity and penetrates the periodontal tissue and is involved in the advanced stage of periodontitis or refractory periodontitis and acute necrotic gingivitis (Non-Patent Document 0013), oral through attachment with other bacteria Tannerella forsythia (Non-Patent Document 0012), which acts as a mediator of my biofilm formation and destroys periodontal tissue to cause periodontitis (Non-Patent Document 0012), produces various toxic factors to break down the host's immune system, loss of alveolar bone, Porphyromonas gingivalis , which can destroy periodontal tissue (Non-Patent Document 0016), is found in acute and chronic periodontitis and attaches to periodontal tissue to cause periodontal tissue destruction Treponema socranskii (Non-Patent Document 0014), Prevotella dentalis ( Prevotella dentalis ) (Non-Patent Document 0011), which has low virulence but at least plays a role in inducing periodontitis (Non-Patent Document 0011), NOD (nucleotide- Binding oligomerization domain) signal transduction contributes to the development of periodontitis, a chronic inflammatory disease, and among them, Eubacterium saphenum, which stimulates NOD1 signaling, Eubacterium saphenum , Eubacterium nodatum , Philifactor allo Filfactor alocis (Non-Patent Document 0015) and Porphyromonas endodontalis (Porphyromonas endodontalis ), which stimulate NOD2 signaling (Non-Patent Document 0015), and Treponema, which cause periodontitis and implant periarthritis, resulting in implant loss. Genus ( Treponema ) (Non-Patent Document 0009), Mycoplasma genus related to periodontal pocket depth (Non-Patent Document 0010), Fretibacterium genus (Non-patented Document 0007), Mogibacterium genus contributing to increased susceptibility to gingivitis and periodontitis in adults (Non-Patent Document 0008) was selected.

As common flora found in healthy groups, Actinomyces genus (Non-Patent Document 0004), which accounts for most of the oral microorganisms and decreases as the ratio of red and orange complexes increases, is found in adults with clean oral hygiene. Oribacterium genus ( Oribacterium ) (Non-Patent Document 0005), Haemophilus parainfluenzae that inhibits the attachment of Porphyromonas gingivalis ( Haemophilus parainfluenzae ) (Non-Patent Document 0006) was selected.

Strains whose results were consistent with non-patent literature, next-generation sequencing (NGS), and clinical samples (oral samples, saliva) of Example 1 were selected as final candidate bacteria, and finally, a total of 4 genera and 9 species as harmful bacteria, A total of 2 genera and 1 species were finally selected as common flora (see Table 4).

Example 3

3.1 Preparation of primers and probes

Primers (see Table 5) and probes (see Table 6) for detecting the periodontitis-related strains finally selected in Example 2 were designed. It is designed to be specific and highly sensitive without mutual interference to detect only the target sequence (16s rRNA) of each strain, and 'Total bacteria' is Gram-positive and Gram-negative microorganisms present in the oral cavity, including selected periodontitis-related bacteria It is designed to detect all of them.

In addition, GC content, melting point, hairpin, hetero-dimer, self-dimer, etc. were considered, and it was confirmed through blast search whether only the corresponding bacteria were specifically detected. The probe used in Real-time PCR is a TaqMan probe type, and the fluorescent dye attached to the probe considers the number of sequences (reads) of next-generation sequencing (NGS) data and wavelengths that do not overlap. Four probe dyes were used.

Tables 5 and 6 are lists of primers and probes designed to bind genus/species specifically.

3.2 Construction of strain plasmid

In order to evaluate the validity of the primers and probes, the target sequences of the strains selected in Table 4 were cloned into plasmids, prepared at a concentration of 10 ⁹ and used as analysis samples.

Example 4

4.1 Singleplex Real-time PCR Conditions

Primers and probes prepared in Example 3 (see Tables 5 and 6) were used at concentrations shown in Table 7. The concentration is the optimal concentration that reacts best.

Composition and PCR conditions for single real-time PCR, 2 μl of forward and reverse primer and probe mixture for each concentration of strain, 2 μl of strain plasmid DNA, 10 μl of 2X Taqman multiplex mastermix (Thermofisher, USA), 3rd 6 μl of sterile water was included to make the final volume 20 μl. Then, it was dispensed into a 96-well plate, and a single real-time polymerase chain reaction (Singleplex Real-time PCR) was performed. A QuantStudio 6 Flex Real-Time PCR System (Thermofisher, USA) was used as a PCR device to perform the PCR reaction, and the reaction was performed at 50 ° C for 2 minutes for the UDG reaction in the contamination prevention step, and then for the inactivation of UDG It was maintained at 95°C for 10 minutes. The steps of PCR amplification were repeated 40 times at 95°C for 15 seconds and 60°C for 30 seconds (see Tables 8 and 9).

4.2 Creation of standard curve using singleplex real-time PCR and evaluation of sensitivity and specificity

In order to confirm that 16 types of periodontitis-related harmful and common bacteria and total bacteria could be detected using the primers, probes and strain plasmids prepared in Example 3, sensitivity evaluation was conducted by applying the conditions presented in 4.1 above. .

Primers and probes for each strain were prepared at the concentrations shown in Table 7, and strain plasmid DNA was serially diluted from 10 ⁹ copy number to 10 ¹ copy number by 1/10, followed by single real-time PCR according to the conditions in Tables 8 and 9. performed. A minimum detection limit and a cycle threshold value for each concentration were measured using a fluorescence amplification plot, and sensitivity and a standard curve were calculated.

After preparing 0.25 x 10 ⁸ copy number of plasmid DNA of each strain (excluding total bacteria) and mixing them all, prepare primers and probes of the 16 strains to be identified at optimal concentrations (see Table 7), and then add one set each. Real-time PCR was performed. 16 μl of the PCR product after the experiment was loaded onto a 5% agarose gel, electrophoresis was performed, and specificity was confirmed (see FIG. 1).

As a result of the experiment, it was confirmed that the primers and probes prepared for 16 types of periodontitis-related harmful and common bacteria and total bacteria detected only the target strain, and the plasmid DNA for each strain was detected at 10 ¹ ~ 10 ² copy number (Table 10 and 11), it was confirmed that the slope was close to -3.33 and was within the ideal qPCR efficiency range (90 to 110%). In addition, it was confirmed that the R ² value of the standard curve for each strain was very close to 1 (about 0.99), indicating that the reliability was very high (see FIGS. 2 to 17). It was confirmed that the primers and probes prepared through the experiment could quantitatively detect and qualitatively distinguish each strain.

4.3 Simultaneous multiplex real-time PCR conditions

By mixing primers and probes (5 sets) for a total of 17 strains, including total bacteria, simultaneous multiplex real-time PCR experiments were performed to confirm that multiple bacteria could be simultaneously detected in a single reaction. Each component of the composition of multiplex real-time PCR consists of 2 forward and reverse primers and probe mixtures of strains corresponding to each set (A set 4 types, B set 4 types, C set 3 types, D set 4 types, E set 2 types) μl, the DNA of the strain plasmid corresponding to each set (A, B, D set 8 μl, C set 6 μl, E set 4 μl) and 10 μl of 2X Taqman multiplex mastermix (Thermofisher, USA), and finally the same volume Including tertiary sterilized water (C set 2 μl, E set 4 μl) to make a total of 20 μl (see Table 12).

Then, it was dispensed into a 96-well plate, and real-time polymerase chain reaction was performed. Simultaneous multiplex real-time PCR was performed at 50 ° C for 2 minutes for the UDG reaction in the contamination prevention step, the same as for single real-time PCR, and then maintained at 95 ° C for 10 minutes to inactivate UDG. The steps of PCR amplification were repeated 40 times at 95°C for 15 seconds and 60°C for 30 seconds (see Table 13).

4.4 Simultaneous Multiplex Real-time PCR

It was confirmed that the primers and probes prepared in 4.2 above qualitatively and quantitatively detect the strain, and based on this, 5 sets of primers and probes that do not cross-react were constructed and multiple simultaneous real-time PCR was performed as in 4.3 above.

It was confirmed that the primer set mixture worked normally with respect to the plasmid DNA mixture for each strain composed of each set (see FIGS. 18 to 22). In addition, serial dilution was performed on the plasmid DNA for each strain of each set, and a standard curve was constructed based on the Ct value (cycle threshold value) of the sample shown in the result of the fluorescence amplification curve. Confirmed. The slope is close to -3.33, and the R ² value of the standard curve is about 0.99, which is very close to 1, so the reliability is very high (see Figs. 18 to 22), and multiple real-time PCR is performed for a total of 17 strains including total bacteria. When performed, it was confirmed that each bacterium was detected at 10 ¹ to 10 ² copy number in all primer and probe mixtures (see Table 14).

In conclusion, oral microbiome data related to dental caries were produced by next-generation sequencing (NGS) based on clinical research samples, and primers and probes for selected harmful bacteria and common bacteria were produced. Through this, we developed species-specific PCR primers and probes that can amplify and detect up to four target strains in a single reaction using the TaqMan simultaneous multiplex real-time PCR technique.

<110> Helixco inc. <120> Information provision method and kit for diagnosis of periodontal disease in Korean <130> 20210430 <160> 52 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for Actinomyces <400> 1 tgtttttggt gggggatggg 20 <210> 2 <211> 23 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Actinomyces <400> 2 tgcttcactg gcgaaagagg ttt 23 <210> 3 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for Fretibacterium <400> 3 agagatgcgc tggaggaggt 20 <210> 4 <211> 26 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Fretibacterium <400> 4 acttccttct tcccacataa aagaac 26 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for Prevotella dentalis <400> 5 tcaatgggcg taagcctgaa 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Prevotella dentalis <400> 6 cggaccttcc gtattaccgc 20 <210> 7 <211> 26 <212> DNA <213> artificial sequence <220> <223> Forward primer for Tannerella forsythia <400> 7 ttacagggga ataaaatgag atacgt 26 <210> 8 <211> 22 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Tannerella forsythia <400> 8 gcattccgcc tacttcatct at 22 <210> 9 <211> 24 <212> DNA <213> artificial sequence <220> <223> Forward primer for Eubacterium nodatum <400> 9 gagcgagaaa tcacttaaag aagc 24 <210> 10 <211> 24 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Eubacterium nodatum <400> 10 aatcgaaatt tctctcggct atcc 24 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for Mogibacterium <400> 11 acggtacctt aggagcaagc 20 <210> 12 <211> 26 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Mogibacterium <400> 12 caagtagagg ttgagcctct aaatta 26 <210> 13 <211> 24 <212> DNA <213> artificial sequence <220> <223> Forward primer for Porphyromonas endodontalis <400> 13 gattgtaaac ttcttttgta gagg 24 <210> 14 <211> 21 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Porphyromonas endodontalis <400> 14 aattccggat aacgctcgta t 21 <210> 15 <211> 25 <212> DNA <213> artificial sequence <220> <223> Forward primer for Treponema <400> 15 agctaagaat attccgcaat ggacg 25 <210> 16 <211> 24 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Treponema <400> 16 tctcaccctt attcttcatc aaca 24 <210> 17 <211> 25 <212> DNA <213> artificial sequence <220> <223> Forward primer for Filifactor alocis <400> 17 ggcattcgtg tcgtaaaact ctgta 25 <210> 18 <211> 23 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Filifactor alocis <400> 18 actaacttgt taaaccacct gcg 23 <210> 19 <211> 20 <212> DNA <213> artificial sequence <220> <223> Forward primer for Treponema denticola <400> 19 tggttggtga ggtaaaggcc 20 <210> 20 <211> 23 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Treponema denticola <400> 20 attagccggg gcttattcgc atg 23 <210> 21 <211> 22 <212> DNA <213> artificial sequence <220> <223> Forward primer for Treponema socranskii <400> 21 gggaggcagc aggtaagaat at 22 <210> 22 <211> 24 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Treponema socranskii <400> 22 ggcatttcct cctacactta ttcc 24 <210> 23 <211> 23 <212> DNA <213> artificial sequence <220> <223> Forward primer for Eubacterium saphenum <400> 23 tgagacacgg tccaaactct acg 23 <210> 24 <211> 25 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Eubacterium saphenum <400> 24 tagtagacca cctacgcact cttta 25 <210> 25 <211> 21 <212> DNA <213> artificial sequence <220> <223> Forward primer for Haemophilus parainfluenzae <400> 25 tcaccaagcc gacgatctct a 21 <210> 26 <211> 23 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Haemophilus parainfluenzae <400> 26 ctttacgccc agttattccg att 23 <210> 27 <211> 22 <212> DNA <213> artificial sequence <220> <223> Forward primer for Oribacterium <400> 27 agcctaccaa agcaacgatc ag 22 <210> 28 <211> 21 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Oribacterium <400> 28 tgtcattatc ttccctgctg a 21 <210> 29 <211> 21 <212> DNA <213> artificial sequence <220> <223> Forward primer for Porphyromonas gingivalis <400> 29 tgcaacttgc cttacagagg g 21 <210> 30 <211> 20 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Porphyromonas gingivalis <400> 30 actcgtatcg cccgttattc 20 <210> 31 <211> 22 <212> DNA <213> artificial sequence <220> <223> Forward primer for Mycoplasma <400> 31 atattccaca atgagcgaaa gc 22 <210> 32 <211> 24 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Mycoplasma <400> 32 tgcatttcct caactaagtg ttct 24 <210> 33 <211> 24 <212> DNA <213> artificial sequence <220> <223> Forward primer for Total bacteria <400> 33 tggagcatgt ggtttaattc gaag 24 <210> 34 <211> 20 <212> DNA <213> artificial sequence <220> <223> Reverse primer for Total bacteria <400> 34 tgcgggactt aacccaacat 20 <210> 35 <211> 26 <212> DNA <213> artificial sequence <220> <223> probe for actinomyces <400> 35 tggacggtca cactgggact gagaca 26 <210> 36 <211> 26 <212> DNA <213> artificial sequence <220> <223> Probe for Fretibacterium <400> 36 caatgggagg aatcctgacc cagcga 26 <210> 37 <211> 25 <212> DNA <213> artificial sequence <220> <223> Probe for Prevotella dentalis <400> 37 atacggggat aaagtgcgcc acgtg 25 <210> 38 <211> 27 <212> DNA <213> artificial sequence <220> <223> Probe for Tannerella forsythia <400> 38 ccttgtgaat aagcatcggc taactcc 27 <210> 39 <211> 27 <212> DNA <213> artificial sequence <220> <223> Probe for Eubacterium nodatum <400> 39 ggtagactta agagatggag agcagcg 27 <210> 40 <211> 26 <212> DNA <213> artificial sequence <220> <223> Probe for Mogibacterium <400> 40 gtgcgtaggt ggttacctaa gcgcaa 26 <210> 41 <211> 25 <212> DNA <213> artificial sequence <220> <223> Probe for Porphyromonas endodontalis <400> 41 gtagctgaga tgcatgtact ctacg 25 <210> 42 <211> 22 <212> DNA <213> artificial sequence <220> <223> probe for treponema <400> 42 cgccgcgtgg atgaagaagg ct 22 <210> 43 <211> 25 <212> DNA <213> artificial sequence <220> <223> Probe for Filifactor alocis <400> 43 cagtacccta aaagaaagcc ccggc 25 <210> 44 <211> 23 <212> DNA <213> artificial sequence <220> <223> Probe for Treponema denticola <400> 44 acggagcgac gccgtgtgaa tga 23 <210> 45 <211> 25 <212> DNA <213> artificial sequence <220> <223> Probe for Treponema socranskii <400> 45 tgaacgaaga aggccggaag gttgt 25 <210> 46 <211> 23 <212> DNA <213> artificial sequence <220> <223> Probe for Eubacterium saphenum <400> 46 cgccgcgtga ggtatgaagg cct 23 <210> 47 <211> 30 <212> DNA <213> artificial sequence <220> <223> Probe for Haemophilus parainfluenzae <400> 47 agtttaatag actaggtgat tgacgttaac 30 <210> 48 <211> 26 <212> DNA <213> artificial sequence <220> <223> Probe for Oribacterium <400> 48 cgccgcgtga gtgaagaagt atttcg 26 <210> 49 <211> 32 <212> DNA <213> artificial sequence <220> <223> Probe for Porphyromonas gingivalis <400> 49 agctgtaaga taggcatgcg tcccattagc ta 32 <210> 50 <211> 24 <212> DNA <213> artificial sequence <220> <223> Probe for Mycoplasma <400> 50 cacgatgaag gccctcgggt tgta 24 <210> 51 <211> 24 <212> DNA <213> artificial sequence <220> <223> Probe for Gram positive bacteria <400> 51 caggtggtgc atggttgtcg tcag 24 <210> 52 <211> 22 <212> DNA <213> artificial sequence <220> <223> Probe for Gram negative bacteria <400> 52 acaggtgctg catggctgtc gt 22

Claims (7)

In a sample derived from the oral cavity of the subject,
as harmful bacteria
A microorganism of the genus Fretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36;
Prevotella dentalis ( Prevotella dentalis );
Tannerella Forsythia ( Tannerella forsythia );
Eubacterium nodatum ;
A microorganism of the genus Mogibacterium detected by PCR using the primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40;
Porphyromonas endodontalis ( Porphyromonas endodontalis );
A microorganism of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42;
Filifactor alocis ;
Treponema denticola ;
Treponema socranskii ;
Eubacterium saphenum ;
Porphyromonas gingivalis ( Porphyromonas gingivalis ); and
The primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50 detected by PCR using Mycoplasma ( Mycoplasma ) genus microorganisms; detecting;
As a normal flora present in healthy subjects
A microorganism of the genus Actinomyces detected by PCR using the primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the probe of SEQ ID NO: 35;
Haemophilus parainfluenzae ; and
Primers of SEQ ID NO: 27, primers of SEQ ID NO: 28, and microorganisms of the genus Oribacterium detected by PCR using the primers of SEQ ID NO: 28 and the probe of SEQ ID NO: 48; Method for providing information for diagnosing periodontal disease in Koreans. According to claim 1,
The primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the primers of SEQ ID NO: 2 and SEQ ID NO: 35 for detection of Actinomyces microorganisms detected by PCR using the primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the probe of SEQ ID NO: 35 PCR using probes;
The primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the primers of SEQ ID NO: 4 and SEQ ID NO: 36 for detection of microorganisms of the genus Pretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36 PCR using probes;
PCR using the primers of SEQ ID NO: 5, the primers of SEQ ID NO: 6, and the probe of SEQ ID NO: 37 for detection of the Prevotella dentalis;
PCR using the primers of SEQ ID NO: 7, the primers of SEQ ID NO: 8, and the probe of SEQ ID NO: 38 for the detection of the Tanerella forsythia;
PCR using the primers of SEQ ID NO: 9, the primers of SEQ ID NO: 10, and the probe of SEQ ID NO: 39 for detection of the Eubacterium nodatum;
The primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40 are used to detect microorganisms of the genus Mosquitobacterium detected by PCR using the primer of SEQ ID NO: 11, the primer of SEQ ID NO: 12, and the probe of SEQ ID NO: 40 PCR using;
PCR using the primers of SEQ ID NO: 13, the primers of SEQ ID NO: 14, and the probe of SEQ ID NO: 41 for detection of the Porphyromonas endodonthalis;
Primers of SEQ ID NO: 15, primers of SEQ ID NO: 16, and probes of SEQ ID NO: 42 for detection of microorganisms of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42 PCR using;
PCR using the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43 for detection of the Philifactor allosis;
PCR using the primers of SEQ ID NO: 19, the primers of SEQ ID NO: 20, and the probe of SEQ ID NO: 44 for detection of the Treponema denticola;
PCR using the primers of SEQ ID NO: 21, the primers of SEQ ID NO: 22, and the probe of SEQ ID NO: 45 for detection of the Treponema sokranskii;
PCR using the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46 for detection of the Eubacterium sapenum;
PCR using the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47 for detection of the Haemophilus parainfluenza;
The primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probes of SEQ ID NO: 28 and the probes of SEQ ID NO: 28 for detection of microorganisms of the genus Orybacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48 PCR using;
PCR using the primers of SEQ ID NO: 29, the primers of SEQ ID NO: 30, and the probe of SEQ ID NO: 49 for detection of the Porphyromonas gingivalis;
The primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50 were used to detect the microorganisms of the genus Mycoplasma detected by PCR using the primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50. A method for providing information for diagnosing periodontal disease in Koreans by performing a PCR; used. According to claim 2,
PCR for detecting microorganisms of the genus Actinomyces detected by PCR using the primers of SEQ ID NO: 1, the primers of SEQ ID NO: 2, and the probe of SEQ ID NO: 35; PCR for detecting microorganisms of the genus Pretibacterium detected by PCR using the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36; PCR for detection of the Prevotella dentalis; And PCR for detection of the Tanerella fositia; carried out in one reaction solution,
PCR for detection of the Eubacterium nodatum; PCR for detecting microorganisms of the genus Mosquitobacterium detected by PCR using the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40; PCR for detection of the Porphyromonas endodonthalis; and PCR for detecting microorganisms of the genus Treponema detected by PCR using the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42; carried out in one reaction solution,
PCR for detection of the Philifactor allosis; PCR for detection of the Treponema denticola; And PCR for detection of Treponema sokranskii; carried out in one reaction solution,
PCR for detection of the Eubacterium sapenum; PCR for detection of the Haemophilus parainfluenza; PCR for detecting microorganisms of the genus Oribacterium detected by PCR using the primers of SEQ ID NO: 27, the primers of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; and PCR for detection of Porphyromonas gingivalis; a method for providing information for diagnosing periodontal disease in Koreans by performing the PCR in one reaction solution. According to claim 3,
PCR using the primers of SEQ ID NO: 33, the primers of SEQ ID NO: 34, and the probe of SEQ ID NO: 51 for detection of Gram-positive microorganisms; and
PCR using the primers of SEQ ID NO: 33, the primers of SEQ ID NO: 34, and the probe of SEQ ID NO: 52 for detection of Gram-negative microorganisms;
PCR for detecting microorganisms of the genus Mycoplasma detected by PCR using the primers of SEQ ID NO: 31, the primers of SEQ ID NO: 32, and the probe of SEQ ID NO: 50; PCR for detection of the Gram-positive microorganism; And PCR for detection of the gram-negative microorganisms; performed in one reaction solution, information providing method for diagnosing periodontal disease in Koreans. For the detection of harmful bacteria
a second primer-probe set comprising the primers of SEQ ID NO: 3, the primers of SEQ ID NO: 4, and the probe of SEQ ID NO: 36;
a third primer-probe set comprising the primers of SEQ ID NO: 5, the primers of SEQ ID NO: 6, and the probe of SEQ ID NO: 37;
a fourth primer-probe set comprising the primers of SEQ ID NO: 7, the primers of SEQ ID NO: 8, and the probe of SEQ ID NO: 38;
a fifth primer-probe set including the primer of SEQ ID NO: 9, the primer of SEQ ID NO: 10, and the probe of SEQ ID NO: 39;
a sixth primer-probe set comprising the primers of SEQ ID NO: 11, the primers of SEQ ID NO: 12, and the probe of SEQ ID NO: 40;
a seventh primer-probe set comprising the primers of SEQ ID NO: 13, the primers of SEQ ID NO: 14, and the probe of SEQ ID NO: 41;
an eighth primer-probe set comprising the primers of SEQ ID NO: 15, the primers of SEQ ID NO: 16, and the probe of SEQ ID NO: 42;
a ninth primer-probe set comprising the primers of SEQ ID NO: 17, the primers of SEQ ID NO: 18, and the probe of SEQ ID NO: 43;
a 10th primer-probe set comprising the primer of SEQ ID NO: 19, the primer of SEQ ID NO: 20, and the probe of SEQ ID NO: 44;
an 11th primer-probe set comprising the primer of SEQ ID NO: 21, the primer of SEQ ID NO: 22, and the probe of SEQ ID NO: 45;
a twelfth primer-probe set comprising the primers of SEQ ID NO: 23, the primers of SEQ ID NO: 24, and the probe of SEQ ID NO: 46;
a 15th primer-probe set comprising the primer of SEQ ID NO: 29, the primer of SEQ ID NO: 30, and the probe of SEQ ID NO: 49; and
A 16th primer-probe set comprising the primer of SEQ ID NO: 31, the primer of SEQ ID NO: 32, and the probe of SEQ ID NO: 50;
For the detection of flora present in healthy subjects
a first primer-probe set comprising a primer of SEQ ID NO: 1, a primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 35;
a 13th primer-probe set comprising the primers of SEQ ID NO: 25, the primers of SEQ ID NO: 26, and the probe of SEQ ID NO: 47; and
A 14th primer-probe set including the primer of SEQ ID NO: 27, the primer of SEQ ID NO: 28, and the probe of SEQ ID NO: 48; a kit for diagnosing periodontal disease in Koreans, comprising: According to claim 5,
The first primer-probe set, the second primer-probe set, the third primer-probe set, and the fourth primer-probe set are included in one composition,
The fifth primer-probe set, the sixth primer-probe set, the seventh primer-probe set, and the eighth primer-probe set are included in one composition,
The ninth primer-probe set, the 10th primer-probe set, and the 11th primer-probe set are included in one composition,
A kit for diagnosing periodontal disease in Koreans, comprising the 12th primer-probe set, the 13th primer-probe set, the 14th primer-probe set, and the 15th primer-probe set in one composition. According to claim 6,
A 17th primer-probe set comprising the primer of SEQ ID NO: 33, the primer of SEQ ID NO: 34, the probe of SEQ ID NO: 51, and the probe of SEQ ID NO: 52; further comprising,
A kit for diagnosing periodontal disease in Koreans, comprising the 16th primer-probe set and the 17th primer-probe set in one composition.