KR20240017711A - Rapid diagnostic kit of oral microbiota for adult and the method using the same - Google Patents

Abstract

The present invention provides a plurality of strips; A labeled detection reagent that specifically binds to the analyte presented by the oral microorganisms and binds to each strip at different concentrations; and a reaction unit located on the strip and coupled with a detection capture member to which the labeled detection reagent is specifically bound. It provides a simple diagnostic kit and diagnostic method for oral microorganism composition for adults.

Description

Simple diagnostic kit for oral microorganism composition for adults and diagnostic method using the same {RAPID DIAGNOSTIC KIT OF ORAL MICROBIOTA FOR ADULT AND THE METHOD USING THE SAME}

The present invention relates to a bio-diagnostic kit, and more specifically, to a simple diagnostic kit for oral microbial composition for adults that can be used as an index for diagnosis during dental examinations by confirming the composition of oral microorganisms, and to a diagnostic method using the same.

Various pathogenic microorganisms exist in the oral dental plaque, which can cause periodontal disease and dental caries. Conversely, checking the dominance and relative abundance of specific microorganisms can be used as an indicator to determine whether it is difficult to immediately detect with the naked eye or the possibility of developing periodontal disease or dental caries in the future.

In addition, through understanding the composition of oral microorganisms, it can be used as an indicator to determine the oral health status of patients visiting dental hospitals, and can be used as data to preemptively prevent oral diseases that may develop.

Currently, for infants and young children, a total of four oral examinations are conducted within the scope of the national health examination in consideration of oral development. However, in the case of infants and young children, the development of oral structure and dentition is just in progress, so only limited factors are checked mainly through visual examination. Accordingly, in order to further increase the effectiveness of oral examination of infants and young children, it is necessary to propose other validation tests that have a great synergy effect when performed together with visual examination.

In particular, exposure to oral microorganisms in infants and young children and the composition of oral microorganisms are factors that can greatly affect oral health during future growth, so early identification and response are important. Furthermore, in adults, systemic diseases such as diabetes, cardiovascular disease, and rheumatic disease are known to be associated with chronic oral infections. The path through which each disease and oral infection occur are diverse, and confirmation of the distribution of these microorganisms in the oral cavity cannot be used as a direct criterion for direct diagnosis of related diseases, but through the detection of microorganisms that can cause inflammation in the oral cavity. It can be used as an indicator of systemic disease risk.

However, to date, research on devices and methods that can check the composition of oral microorganisms and use them as diagnostic indicators during dental examinations is very minimal, and improvement is urgently needed.

Accordingly, the present inventors came up with the idea to solve the above-mentioned problems, and the purpose is to provide a simple diagnostic kit for adults that can be used as an index for diagnosis during dental examinations by checking the composition of microorganisms in the oral cavity and a diagnostic method using the same.

The technical object of the present invention as described above is achieved by the following means.

(1) Multiple strips; A labeled detection reagent that specifically binds to the analyte presented by the oral microorganisms and binds to each strip at different concentrations; and a reaction unit located on the strip and coupled with a detection capture member to which the labeled detection reagent is specifically bound.

(2) The diagnostic kit in (1) above uses S.mutans, Actinomyces strains, P.gingivalis, and P.intermedia as indicator strains for the screening of dental caries in infants and young children, and tests the antigens presented by each of these cells. A simple diagnostic kit for oral microorganism composition for adults, characterized in that a specifically binding detection reagent is attached to each strip.

(3) The diagnostic kit in (2) above further adds S. mitis as an indicator strain, and a detection reagent that specifically binds to the antigen presented by S. mitis is attached to one of the strips. Simple diagnostic kit for oral microorganism composition for adults.

(4) preparing a plurality of strips in which labeled detection reagents are combined at different concentrations; Loading a sample onto the prepared plurality of strips; A step in which the analyte contained in the sample binds to the labeled detection reagent and moves to the reaction zone on the strip; A step of specifically binding the labeled detection reagent to which the analyte is bound to a detection capture member bound to the reaction unit; and calculating the concentration of the analyte by analyzing the reaction results that occurred in the reaction section according to the marker with different concentration in each strip.

(5) The diagnostic kit in (4) above uses S.mutans, Actinomyces strains, P.gingivalis, and P.intermedia as indicator strains to screen for dental caries in infants and children, and tests the antigens presented by each of these cells. A simple diagnostic method for oral microorganism composition for adults, characterized in that a specifically binding detection reagent is attached to each strip.

(6) The diagnostic kit in (5) above further adds S. mitis as an indicator strain, and a detection reagent that specifically binds to the antigen presented by S. mitis is attached to one of the strips. A simple diagnostic method for oral microorganism composition for adults.

As described above, according to the present invention, the composition of microorganisms in the oral cavity of adults can be used as an indicator to determine the oral health status of patients visiting a dental hospital, and data for preemptively preventing oral diseases that are likely to progress is provided. In that it can be used, it has the effect of contributing to the improvement of national health as well as the development of related industries.

Figure 1 is a schematic diagram of the sample collection device of the present invention and a diagnostic kit including the same.
Figure 2 is a configuration diagram showing an embodiment of the diagnostic kit of the present invention.
Figure 3 is an exemplary configuration diagram of a specimen collection means according to the present invention.

In order to achieve the above object, the diagnostic kit according to the present invention includes a plurality of strips; A labeled detection reagent that specifically binds to the analyte presented by the oral microorganisms and binds to each strip at different concentrations; and a reaction portion located on the strip to which a detection capture member to which the labeled detection reagent is specifically bound is bound.

In addition, the method for analyzing the concentration of an analyte using a diagnostic kit according to the present invention includes the steps of preparing a plurality of strips in which labeled detection reagents are combined at different concentrations; Loading a sample onto the prepared plurality of strips; A step in which the analyte contained in the sample binds to the labeled detection reagent and moves to the reaction zone on the strip; A step of specifically binding the labeled detection reagent to which the analyte is bound to a detection capture member bound to the reaction unit; And calculating the concentration of the analyte by analyzing the reaction results that occurred in the reaction section according to the label with different concentration in each strip.

Figure 1 is a configuration diagram of the sample collection device of the present invention. The sample collection device 40 includes a sample collection means 20 and a sample recovery means 40.

The sample collection means (20) consists of a support unit (10), a sample collection unit (11), and a grip unit (12). The support 10 has a sample collection unit 11 coupled to one end, and a gripping unit 12 is coupled to the other end.

Preferably, the support 10 is made of a flexible plastic material so that the sample collection unit 11 can be easily positioned at the target area when collecting a sample. The sample collection unit 11 is a part for collecting samples growing in the oral cavity, and may be made of, for example, a cotton material.

Preferably, the gripping part 12 is made of a compressible tube (e.g., an air-filled rubber tube) as shown in FIG. 3, and the support 10 is made of a hollow tube, and the connection part of the gripping part and the support. A reverse lever (12a) is installed. Therefore, after placing a sample in the sample collection unit 11, when the gripper 12 is pressed, the air trapped in the gripper is released through the reverse valve 12a, and the released air is in the sample collection unit 11. Make it easy to remove the buried specimen.

“Sample” refers to something that grows in the oral cavity and includes microorganisms, including bacteria and mold, or viruses. The bacteria are preferably Streptococcus series (S. mutans, S.sobrinus, S. mitis, etc.), Actinomyces series (A. gerencseriae, A. viscosus, etc.), and Porphyromonas gingivalis (P. gingivalis). ), and various bacteria that cause oral diseases such as Prevotella intermedia (P. intermedia).

Bacteria subject to analysis may vary depending on age. For example, in the case of infants and young children, screening for dental caries is an important factor, so Mutans groups such as S.mutans and S.sobrinus are set as indicator strains (produced with two types of diagnostic kits). For adults, in addition to S.mutans, it is desirable to include the Actinomyces family, P.gingivalis, an indicator strain of periodontitis, and P.intermedia, an indicator strain of plaque (produced with 4 types of diagnostic kits). In particular, for adults with a history of cardiovascular disease such as infective endocarditis or systemic disease, it is desirable to add S. mitis (produced in a 5-piece kit).

In the above, the sample may be collected differently depending on the age of the subject, the purpose of the examination, and the disease to be diagnosed. When collecting saliva, collect plaque from around the opening of the sublingual gland and submandibular gland on the floor of the mouth, from the buccal sulcus, from the supragingival plaque, from the buccal gingiva and tooth area around the upper and lower jaw, and from the lingual surface of the maxilla or mandibular anterior teeth, for subgingival plaque. It is desirable to collect samples from 4 areas.

The sample recovery means 40 consists of a cover 31 and a sample recovery container 32, and a through hole is formed in the center of the cover 31 so that the sample collection means 10 penetrates into the sample recovery container 32. is inserted. The sample recovery container 32 contains a reagent (e.g., detergent for dissolving cell membrane, surfactant, etc.) or medium for extracting the analyte, and recovers the sample stuck on the sample collection unit 11. It is provided for extracting desired analytes.

The test solution (hereinafter referred to as sample) contained in the sample recovery container 32 is transferred to the sample loading means 50 (e.g., a pipette or a device equipped with a separate injection port that performs a similar function), and simple diagnosis is performed. It is loaded into the kit 200.

Figure 2 is a configuration diagram of the simple diagnostic kit 200 of the present invention. The simple diagnostic kit 200 of the present invention includes at least one strip 100 and a housing that protects it.

The housing includes a sample input unit 150, a cover 160 with a display window 161 for showing test results, and a lower support 170 on which a plurality of strips 100a, 100b,... are fixedly placed in place. do. Each of the strips is for individual diagnosis of specific microorganisms.

Changes occurring in the test lines (T1 to T4) and control lines (C1 to C4) on the development films (103 to 106) constituting the strip (100) can be observed from the outside through the display window (161).

The strip (1) according to the present invention preferably includes a storage pad (101), a label pad (102), a development film (103-106), and an absorption pad (107).

The storage pad 101 is required to have sufficient voids and volume to accommodate the sample. Materials that can be used as such storage pads are preferably low-molecular protein binding materials such as cellulose, polyester, polyurethane, and glass fiber with a pore size of 0.45 to 60 μm.

The analyte (1), which is the target of analysis in the sample, includes any substance having a specific binding site that can be naturally formed or artificially assigned, for example, antigen presenting substance, heptene, antibody, Or it may be a combination thereof. In the present invention, the analyte is preferably a specific antigen presented by oral microorganisms.

In the present invention, extraction of specific antigens for oral microorganisms can be prepared in various ways. For example, using detergents such as sodium deoxycholate, sodium dodecyl sulfate, or sodium decyl sulfate (e.g., US 4,741,999), or osmotic shock (e.g., Dirien zo et al, Infect. & Immun., 47(1), pp. 31-36, 1985), and can be performed using physical or chemical means such as ultrasonic means (e.g., Zambon et al, Infect. & Immun., 41(1), pp. 19-27, 1983). there is. Preferably, it is an extraction method using a cationic surfactant and an anionic surfactant with a high pH composition.

The labeling pad 102 is in contact with the lower end of the developing membranes 103 to 106 for chromatography to form a connecting passage. The labeling pad 102 filters unnecessary components in the sample and includes a detection reagent 110. Examples of materials that can be used as the label pad 102 include polyurethane, polyacetate, cellulose, glass fiber, and nylon with a pore size of 0.45 to 60 μm.

In the present invention, the detection reagent 110 does not require any special limitations, but if the analyte 1 is an antigen presented by oral microorganisms, an antibody that specifically binds to the antigen, and if the analyte 1 is an antibody, the antibody binds to the antigen. It may be protein G, protein A, protein G/A, which are known to be easily binding substances, or various antibodies known to bind well, such as other antibodies such as IgG and IgM.

The antibody used as the detection reagent 110 in the present invention may be a monoclonal antibody or a polyclonal antibody. Monoclonal antibodies can be prepared by the method disclosed in US 4,741,999, and polyclonal antibodies can be prepared using the method disclosed by Zambon et al.

The label 111 attached to the detection reagent 110 includes the following. Examples include catalysts, enzymes (e.g., phosphotases, peroxidase, etc.; more specific examples include basic phosphatase and lamb pepper peroxidase, which are used in combination with enzyme substrates), enzyme substrates (e.g., , nitroblue tetrazolium, 3,5',5,5'-tetranitrobenzidine, 4-methoxy1naphthol, 4chloro1naphthol, 5bromo4chloro3indolylphosphate, and dioxetane as the chemiluminescent enzyme substrate. , and derivatization and their analogs), fluorescent compounds (e.g., fluorescein, phycobiliprotein, rhodamine, etc. and their derivatives and analogs), chemiluminescent compounds, radioactive elements, Metal sol containing nano gold particles, dye sol, particulate latex, color indicator, color matter contained in liposomes, carbon sol and selenium There are similar non-metallic sols.

The detection reagent 110 attached to each strip (100a, 100b,...) is required to specifically bind to the antigen specifically presented by the corresponding microorganism.

The developing films (103 to 106) must have sufficient pores and be able to absorb a significant portion of the sample that has passed through the labeling pad (102). Examples of materials that can serve as such a developing film include nylon, cellulose, polysulfone, polyvinylidene difluoride, cellulose acetate, polyurethane, glass fiber, nitrocellulose, etc.

A detection capture member 120 and a control capture member 130 are attached to the development films 103 to 106, and are preferably attached so that the concentration increases in the direction in which the development progresses, respectively, at the test line (T) and Form a control line (C).

The detection capture member 120 is an unlabeled object that specifically binds to the analyte 1 and captures the analyte, and may be a protein, antigen, or antibody, and is directly or indirectly fixed to the strip.

The control capture member 130 is used to determine whether the kit operates normally, and may be selected from labeled proteins, antigens, antibodies, etc. that specifically bind to the detection reagent.

The absorption pad 12 is attached to the upper end of the development films 103 to 106 and absorbs the sample so that it can be developed on the strip 100.

In the present invention, a pH indicating means (litmus paper, reagent, etc.) 140 is preferably formed at a predetermined position on the strip (e.g., developing film 103) to measure the pH of the sample. At this time, the color change indicated by the pH indicating means is displayed externally through the display window 161.

As shown in FIG. 2, according to a preferred embodiment of the present invention, the higher the concentration of the antigen, which is the analyte 1, in the sample, the more specific the detection capture member 120 attached to the developing membrane 103 to 106. Due to increased binding, the color density becomes darker or more color changes in the test line (T) are observed in the development direction. For example, in the case of strip 100a for S. mutans diagnosis, the color change on the test line in the development direction is 3 spaces, in the case of strip 100b for S. sobrinus diagnosis, 4 spaces, and in the case of strip 100c for P. gingivalis diagnosis, 0 spaces, In the case of 100d of strips for P. intermedia diagnosis, if 1 space is observed, the concentration of each microorganism can be estimated through predetermined pattern analysis and the composition or distribution of microorganisms in the tester's oral cavity can be specified.

As described above, the present invention has been described with reference to preferred embodiments, but those skilled in the art may make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. You will understand that you can do it.

1: Analyte
20: Sample collection means
40: Specimen recovery means
50: sample loading means
100: strip
200: Diagnostic kit

Claims (6)

plural strips; A labeled detection reagent that specifically binds to the analyte presented by the oral microorganisms and binds to each strip at different concentrations; and a reaction unit located on the strip and coupled with a detection capture member to which the labeled detection reagent is specifically bound. According to clause 1,
For the screening of dental caries in infants and young children, S.mutans, Actinomyces strains, P.gingivalis, and P.intermedia are used as indicator strains, and a detection reagent that specifically binds to the antigen presented by each of these cells is used in each strip. A simple diagnostic kit for oral microorganism composition for adults, characterized in that it is attached to. According to clause 2,
A simple diagnostic kit for oral microorganism composition for adults, characterized in that S. mitis is further added as an indicator strain, and a detection reagent that specifically binds to the antigen presented by S. mitis is attached to one of the strips. Preparing a plurality of strips in which labeled detection reagents are combined at different concentrations; Loading a sample onto the prepared plurality of strips; A step in which the analyte contained in the sample binds to the labeled detection reagent and moves to the reaction zone on the strip; A step of specifically binding the labeled detection reagent to which the analyte is bound to a detection capture member bound to the reaction unit; and calculating the concentration of the analyte by analyzing the reaction results that occurred in the reaction section according to the marker with different concentration in each strip. According to clause 4,
For the screening of dental caries in infants and young children, S.mutans, Actinomyces strains, P.gingivalis, and P.intermedia are used as indicator strains, and a detection reagent that specifically binds to the antigen presented by each of these cells is used in each strip. A simple diagnostic method for oral microorganism composition for adults, characterized in that it is attached to. According to clause 5,
A simple diagnostic method for oral microorganism composition for adults, characterized in that S. mitis is further added as an indicator strain, and a detection reagent that specifically binds to the antigen presented by the S. mitis is attached to one of the strips.

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