CN115980368A

CN115980368A - Marker group for periodontitis detection

Info

Publication number: CN115980368A
Application number: CN202310095582.5A
Authority: CN
Inventors: 宋锦璘; 张曦木; 神华尉; 袁徐蕾
Original assignee: Stomatological Hospital of Chongqing Medical University
Current assignee: Stomatological Hospital of Chongqing Medical University
Priority date: 2023-02-08
Filing date: 2023-02-08
Publication date: 2023-04-18
Anticipated expiration: 2043-02-08
Also published as: CN115980368B

Abstract

The invention relates to the field of periodontitis detection, and in particular relates to a marker group for periodontitis detection. The present invention provides a marker set for assessing the periodontitis state of a subject, the marker set comprising: a) Amount of gingival crevicular fluid; b) pH of gingival crevicular fluid; c) Gingival crevicular fluid hemoglobin. The marker set provided by the invention can evaluate the periodontitis state of a subject from multiple dimensions, and accurately and sensitively reflect the development stage and development trend of periodontitis of different subjects.

Description

Marker group for periodontitis detection

Technical Field

The invention relates to the field of periodontitis detection, and in particular relates to a marker group for periodontitis detection.

Background

Periodontitis is one of three diseases of the oral cavity and the sixth most common disease of human, and the worldwide incidence rate is up to 45% -50%. Typical symptoms of periodontitis are destruction of the supporting tissues of the teeth, including bleeding of the gums, gingival atrophy, alveolar bone resorption, and even tooth loosening and loss. Periodontitis is reported to be a major cause of tooth loss in adults. Periodontitis seriously impairs the chewing function of patients, not only limits their food choices and affects their beauty, but also has a great negative impact on their physical and mental health and quality of life. A number of reports have demonstrated that periodontitis is associated with a number of systemic diseases, such as cardiovascular and cerebrovascular diseases, diabetes, cancer of the digestive tract, and the like. In addition, the course of periodontitis can last for decades, placing a considerable burden on individuals and society. Therefore, early and accurate diagnosis of periodontitis is critical from an oral and overall health perspective.

The current clinical diagnosis of periodontitis relies mainly on chair-side clinical examination and imaging examination by the dentist. First, the dentist will ask the patient for medical history, such as systemic, oral, periodontal and oral hygiene habits, and family history. Subsequently, the stomatologist will perform an examination of the periodontal tissue within the patient's mouth, such as examining the patient's oral hygiene (e.g., gingival index, bleeding index, etc.) through plaque staining tests and routine visual judgment; judging whether the teeth of the patient have periodontal pockets, probing bleeding, root bifurcation lesion and the like through periodontal probing; checking the looseness of teeth by using dental forceps, checking whether occlusal interference exists by using occlusal paper, and the like; in addition, the alveolar bone absorption condition can be visually displayed through X-ray images. As can be seen, the above examination is not only time-consuming and requires a high degree of patient compliance, but also has a highly subjective diagnosis result, which is not conducive to objective assessment of the progression of periodontitis in patients (e.g., most patients with periodontitis are not treated in a fixed medical location, and even if treated in the same medical location, the dentists who treat each treatment may be different).

Although some auxiliary periodontal inspection methods (such as pressure sensitive probes, dentition dynamics instruments, etc.) exist at present, most of the auxiliary inspection methods are tedious, time-consuming, and not conducive to rapid chair-side completion by the dentist, and thus are not commonly used.

Disclosure of Invention

In a first aspect, the present invention provides the use of a marker panel in the manufacture of a kit for assessing the periodontitis state in a subject, wherein the marker panel comprises:

a) Amount of gingival crevicular fluid;

b) pH of gingival crevicular fluid;

c) Gingival crevicular hemoglobin;

the kit comprises a detection reagent for detecting the value of the set of markers in a gingival crevicular fluid sample of the subject; the periodontitis state includes a stage of periodontitis progression and/or a trend of periodontitis progression.

In some embodiments, the marker set further comprises a porphyromonas gingivalis-related marker comprising an amount of porphyromonas gingivalis and gingipain K.

In some embodiments, the set of markers further comprises periodontitis-associated biochemical markers comprising matrix metalloproteinase-8 (MMP-8), interleukin-6 (IL-6), interleukin-1 β (IL-1 β), 8-hydroxydeoxyguanosine (8-OHdG), and Malondialdehyde (MDA).

In some embodiments, the subject has a propensity to develop periodontitis.

In some embodiments, the periodontitis progression comprises a worsening of periodontitis or a amelioration of periodontitis.

In some embodiments, the subject is assessed as having a strong positive trend for the progression of periodontitis when all of the markers in the marker set are above a reference value; (ii) when one or more markers in the marker set is above the reference value, the subject is assessed as having a positive trend for the progression of periodontitis; (ii) the subject is assessed as having a negative trend of progression of the periodontitis when none of the markers in the marker set is above the reference value; the subject is assessed as having a trend of an improvement in the periodontitis when none of the markers in the marker set are above the reference value and one or more markers are below the reference value.

In some embodiments, the stages of periodontitis progression include stage zero periodontitis, stage one periodontitis, and stage two periodontitis.

In some embodiments, the periodontitis progression stages include a periodontal health stage, a mild periodontitis stage, and a severe periodontitis stage.

In some embodiments, the subject comprises a periodontitis patient.

In some embodiments, the detection reagent is an aptamer that specifically recognizes the label.

In a second aspect, the present invention provides a marker panel for assessing the periodontitis state including periodontitis progression stage and/or periodontitis trend in a subject, the marker panel comprising:

a) Amount of gingival crevicular fluid;

b) pH of gingival crevicular fluid;

c) Gingival crevicular fluid hemoglobin.

In some embodiments, the periodontitis progression stage comprises a periodontitis zeroth stage, a periodontitis first stage, and a periodontitis second stage.

Compared with the prior art, the invention has the beneficial effects that:

although the conventional clinical examination method can determine the site, extent and type of periodontitis lesion, it cannot reflect the state change of periodontitis. The onset and development of periodontitis is not a simple, persistent process, and may change in development in the acute phase or relapse after treatment. Even if the clinical symptoms are the same, different patients may respond to different degrees of periodontitis disease and trends in periodontitis progression. In addition, conventional clinical examination methods have drawbacks in sensitivity, for example, patients may have occult bleeding in gingival crevicular fluid before clinically detecting positive exploratory bleeding, which is detrimental to early detection, early diagnosis, and early treatment of periodontitis. The marker set provided by the invention can evaluate the periodontitis state of a subject from multiple dimensions, and accurately and sensitively reflect the development stage and development trend of periodontitis of different subjects.

Currently, conventional forms of periodontitis treatment include basic treatments (including supragingival scaling, subgingival scaling, and root planing), drug treatments (topical and systemic use of antibiotics), and periodontal surgery treatments. The prior art often uses a single dimension (e.g., hemoglobin) to diagnose whether a subject has periodontitis, and the diagnosis based on the single dimension does not prompt the dentist or the subject himself to select an appropriate treatment. As a result, the subject may also be treated with a broad spectrum antibiotic, which may be less than optimal, and may be harmful to probiotics, disturb the oral micro-ecology, or cause the proliferation of resistant bacteria. The evaluation result obtained based on the marker set provided by the invention can comprehensively and truly reflect the periodontitis state of the subject, and the dentist or the subject can be prompted to select a more appropriate treatment scheme through the value of a specific marker so as to realize the targeted treatment on specific types of microorganisms and specific types of disease states.

Based on the marker set, the marker set provided by the invention is not only beneficial to realizing dynamic, personalized and long-term monitoring on the periodontitis development state of a subject, but also beneficial to a clinician to optimize a targeted personalized treatment scheme and effectively evaluate the curative effect of the treatment scheme.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale. It is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.

Fig. 1 is a schematic structural diagram of an extraction device in a third exemplary embodiment of the present invention;

FIG. 2 is a schematic view of the internal structure of the extraction apparatus shown in FIG. 1;

FIG. 3 is a schematic view of the internal structure of the mixing chamber of the extraction apparatus shown in FIG. 1;

FIG. 4 is a schematic view of the stand-alone storage unit of the extraction device of FIG. 1 after extraction of gingival crevicular fluid;

FIG. 5 is a schematic view of the extraction device of FIG. 1 in use;

FIG. 6 is a schematic structural diagram of an extracting apparatus according to a fourth exemplary embodiment of the present invention;

FIG. 7 is a first schematic view of the internal structure of the extraction device shown in FIG. 6;

FIG. 8 is a second schematic view of the internal structure of the extraction device shown in FIG. 6;

FIG. 9 is a schematic view of the extraction device of FIG. 6 in use;

FIG. 10a is a first structural schematic view of the reverse side of a test card in an exemplary embodiment of the invention;

FIG. 10b is a schematic diagram of the front side of the test card in an exemplary embodiment of the invention;

FIG. 10c is a second schematic view of the reverse side of the test card in an exemplary embodiment of the invention;

FIG. 11 is a schematic view of a test card and a test instrument for use with the test card in an exemplary embodiment of the invention;

FIG. 12 is a first schematic view of the test card of FIG. 11 in mating engagement with a test instrument;

FIG. 13 is a second schematic view of the test card of FIG. 11 in mating engagement with a test instrument.

Wherein 10 is an extraction tube, 11 is a capillary channel, 12 is an extraction end, 13 is an air pressure balance tube, 14 is a scale mark, and 15 is a breaking structure; 20 is a mixing cavity, 21 is an accommodating space, and 22 is a liquid outlet; 30 is a pushing part, 31 is a first end, and 32 is a second end; 40 is a first storage chamber, 41 is a second opening, and 42 is a first storage space; 50 is the second storage chamber, 51 is the air pressure balance port, 52 is the second storage space, 53 is the first channel, 60 is the body, 61 is the sample injection port, 62 is the first detection zone, 63 is the second channel.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Herein, the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience in describing and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

As used herein, unless otherwise expressly specified or limited, the terms "mounted," "disposed," "connected," and the like are to be construed broadly, such that the terms "connected," or "connected," as used herein, may be fixedly connected, detachably connected, or integrally connected; they may be mechanically coupled, directly coupled, indirectly coupled through intervening media, or interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

Herein "and/or" includes any and all combinations of one or more of the associated listed items.

By "plurality" herein is meant two or more, i.e. it includes two, three, four, five, etc.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.

As used in this specification, the term "about" typically means +/-5% of the stated value, more typically +/-4% of the stated value, more typically +/-3% of the stated value, more typically +/-2% of the stated value, even more typically +/-1% of the stated value, and even more typically +/-0.5% of the stated value.

In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of the range 1 to 6 should be read as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.

The present invention provides a marker set for assessing the periodontitis state of a subject, the marker set comprising:

a) Amount of gingival crevicular fluid;

b) pH of gingival crevicular fluid;

c) Gingival crevicular fluid hemoglobin.

In some embodiments, the marker panel further comprises a porphyromonas gingivalis-related marker comprising an amount of porphyromonas gingivalis and a gingipain K (KGP).

As used herein, the term "marker" or "biomarker" refers to an indicator that is capable of reflecting a correlation with a particular physical condition or state. In this context, a "biomarker" may be a biomacromolecule (e.g. protein, nucleic acid), a small molecule (e.g. compound), or any information or parameter (e.g. pH) of a biological sample. Thus, as used herein, "information on a marker" or "value on a marker" refers to any information associated with the marker. As a non-limiting example, if the biomarker is pH, the value of its marker (e.g., information about the marker) may be 7.1 for a biological sample having a pH of 7.1.

As used herein, the term "sample" or "biological sample" or "specimen" refers to a biological material isolated from a subject. The sample may comprise any biological material suitable for detecting the desired marker, and may comprise cellular and/or non-cellular material from the subject. The sample may be isolated from any suitable biological tissue or fluid. In the present invention, the sample is gingival crevicular fluid. The sample may be pretreated prior to actual detection by methods including filtration, distillation, extraction, concentration, dilution, inactivation of interfering components, addition of reagents, and the like.

As used herein, the terms "subject," "individual," and "patient" are used interchangeably to refer to the mammal from which a sample is taken, unless otherwise specified. A subject, individual or patient may have periodontitis, be at risk of developing periodontitis or is suspected of having a propensity to develop periodontitis. In some embodiments, a typical subject comprises a human patient susceptible to, suffering from, or having suffered from one or more periodontitis. Thus, in some embodiments, the marker sets provided herein can be used to assess the stage of progression (e.g., having mild periodontitis or severe periodontitis) and/or the trend of progression (e.g., worsening or improvement) of periodontitis in a human patient already having periodontitis.

As used herein, the term "predisposition," e.g., "having a predisposition to develop periodontitis," refers to a reasonable medical probability of an event (e.g., periodontitis occurrence or recurrence). The term "predisposition" also includes the frequency with which such events may occur before, after or during the duration of treatment.

A "reference value" for a marker may be an absolute or relative value or concentration of the marker, the presence or absence of the marker, a range of values or concentrations of the marker, the lowest and/or highest values or concentrations of the marker, a mean value or concentration of the marker and/or a median value or concentration of the marker. The reference value and the value of the marker of the subject may vary depending on the particular technique used to determine the marker in the biological sample.

The value of the marker of the subject can be compared to its reference value for reference using a variety of means, such as simple comparison, statistical analysis (e.g., t-test, wilcoxon rank sum test). In some embodiments, the comparison may be accomplished by a manual and/or automated system (e.g., test card, test instrument, provided below). In some embodiments, a value of one or more markers of a subject that is 5%, 10%, 15%, 20% or more above its reference value can be considered an increase in the value of one or more markers of the subject.

The "value" or "level" of one or more markers (or indicators) refers to the absolute (quantitative) or relative (qualitative) value or concentration of the biomarker in the sample.

Gingival crevicular fluid is the fluid that infiltrates the gingival sulcus from the gingival connective tissue into the gingival sulcus by the epithelium and associated epithelium, and is primarily derived from serum and other components from adjacent periodontal tissues and bacteria. The gingival crevicular fluid from the subject may be collected and the amount of gingival crevicular fluid read using collection methods known in the art (e.g., capillary and filter paper strip methods). Preferably, a gingival crevicular fluid sample from a subject may be collected using an extraction device provided by the present invention.

The collected gingival crevicular fluid sample may be analyzed using a suitable method to detect the value of each marker in the biological sample.

Each label may be detected by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. Exemplary suitable methods include chromatography (e.g., liquid Chromatography (LC), high Performance Liquid Chromatography (HPLC), gas Chromatography (GC)), mass spectrometry (e.g., mass Spectrometry (MS), tandem mass spectrometry (MS/MS 2)), combinatorial methods (gas chromatography/mass spectrometry (GC-MS), liquid chromatography/mass spectrometry (LC-MS), ultra-high performance liquid chromatography/tandem mass spectrometry (UHLC/MS 2), and gas chromatography/tandem mass spectrometry (GC/MS 2)), immunochemical techniques such as enzyme-linked immunosorbent assay (ELISA), and combinations thereof, refractive index spectrometry (RI), ultraviolet spectrometry (UV), fluorescence analysis, radiochemical analysis, near-infrared spectrometry (near-IR), nuclear magnetic resonance spectrometry (NMR), light scattering analysis (LS). Additionally, in some embodiments, the value of one or more labels may also be measured by indirect measurement, such as measuring the value of a certain compound (or compounds) that correlates with the value of the label desired to be measured.

In some embodiments, preferred detection methods for some markers of the invention (e.g., gingival crevicular hemoglobin, amount of Porphyromonas gingivalis, matrix metalloproteinase-8, interleukin-6, interleukin-1 β, 8-hydroxydeoxyguanosine, malondialdehyde, and Gum proteinase K) include immunoassays or aptamer-based assays. Such immunoassays or aptamer-based assays are conventional and well known in the art, involving the use of reagents (e.g., antibodies or aptamers) specific for the substance of interest. An agent that "specifically binds" a target substance (e.g., an antibody or aptamer) is a term well known in the art, and methods for determining specific binding are also well known in the art. In some embodiments, the invention measures the protein concentration of the marker. Protein concentration is typically measured in mass per volume, e.g., mg/ml, μ g/ml, and pg/ml.

In some embodiments, the pH of the gingival crevicular fluid, a marker of the present invention, can be measured by a pH indicator method (e.g., methyl red and bromothymol blue), and can also be measured by a pH paper (e.g., precision pH paper) or a pH meter.

As used herein, a "detection reagent" is a reagent or compound that specifically (or selectively) binds, interacts with, or detects a label. In some embodiments, the detection reagent comprises an antibody, a polyclonal antibody, or a monoclonal antibody. In some embodiments, the detection reagent comprises a complex formed with an antibody, polyclonal antibody, or monoclonal antibody (e.g., a fluorescent particle immobilized with an antibody). In some embodiments, the detection reagent comprises an aptamer.

After determining the value of one or more markers in a biological sample from a subject, the value is compared to a reference value to which it is referenced. The reference value may comprise a fixed reference value for each marker and/or a reference value derived from the person himself. When a subject first evaluates the periodontitis development status by the marker set provided by the present invention, the values of all markers in the marker set of the subject are usually compared with corresponding fixed reference values, so as to evaluate the periodontitis development stage of the subject: (ii) if the values of all markers of the subject belong to the zeroth phase reference value, the subject is assessed as periodontal health; if the subject's values of some (or all) of the markers belong to the first stage reference value and another part (or none) belongs to the zeroth stage reference value, the subject is evaluated as a patient with mild periodontitis (or at the first stage of development of periodontitis); if the subject's values for some (or all) of the markers belong to the second stage reference value and another part (or none) belongs to the zeroth or first stage reference value, the subject is assessed as a patient with severe periodontitis (or at the second stage of periodontitis development). When the subject has been assessed at least once for their periodontitis progression status by the marker sets provided herein, the subject's value of each marker can be compared to its last (or optional one or more) test results to further assess the subject's periodontitis progression status.

Based on the above-obtained results of the stage of periodontitis development, the subject's trend of periodontitis development (particularly for subjects in the first and second stages of periodontitis) can be further evaluated by comparing the values of all the markers of the subject with their own reference values. If the values of all markers in the subject's marker panel are not significantly increased (i.e., not higher) as compared to their reference value derived from the subject (e.g., are the same as, substantially the same as, not statistically different from, or are within a given range of the reference value, etc.) then the subject is assessed as having a lower risk of progression of periodontitis (which may be considered not to be worsening, i.e., a negative trend of progression of periodontitis) for a period of time from the date the biological sample was taken; if an increase in the value of one or more markers in the marker panel occurs compared to their own reference value, the subject is assessed as having a certain risk of periodontitis exacerbation (i.e. a positive trend of periodontitis exacerbation) for a period of time from the date the biological sample was taken. In particular, the values of all the markers of the marker panel are elevated compared to their own reference values, to which they are referred, and the subject is assessed as having a very high risk of periodontitis exacerbation (i.e. a strong positive trend of periodontitis exacerbation) for a period of time from the date the biological sample was taken. Of course, if the values of most or all of the markers in the marker panel are decreased (and not significantly increased for other markers) compared to their reference values, then the subject is assessed as having a trend toward improved periodontitis over a period of time from the date the biological sample was taken.

The development of periodontitis is not a continuous process, and there are both changes in development in the acute phase and the possibility of recurrence after treatment. Patients with the same clinical symptoms may respond to different degrees of periodontitis disease. The marker set provided by the invention can accurately and sensitively evaluate the periodontitis development state (such as periodontitis development stage and periodontitis development trend) of a subject from multiple dimensions, is not only beneficial to stomatologists to evaluate, diagnose and monitor (namely, clinical monitoring) the periodontal health condition of patients, but also beneficial to patients to simply and accurately evaluate, diagnose and monitor (namely, self-monitoring) the periodontal health condition of the patients, and avoids the medical resource extrusion, and is especially suitable for the patients suffering from periodontitis or being easy to relapse of periodontitis or having the risk of periodontitis deterioration. The marker set provided by the invention can evaluate the change of the periodontitis development state of a subject early (for example, patients with mild periodontitis or periodontal health populations at risk of periodontitis deterioration can be identified more accurately) and provide early warning (for example, oral care measures need to be taken timely, oral hygiene habits need to be changed, OTC medicines need to be taken, and the like), so that the periodontitis deterioration of the populations can be prevented to a certain extent. Of course, patients who are evaluated by the marker panel of the present invention as patients with severe periodontitis or patients with mild periodontitis and at risk of periodontitis deterioration will require immediate medical attention to prevent further deterioration.

Because the marker set provided by the invention can evaluate the periodontitis state of the subject from multiple dimensions, an oral doctor or the subject himself can select a more targeted treatment scheme (for example, antibacterial treatment aiming at the Porphyromonas gingivalis can be selected if the value of the related marker of the Porphyromonas gingivalis in the detection result is obviously increased), or further carry out metagenome sequencing or 16sRNA sequencing under the condition of abnormal Porphyromonas gingivalis, so that the oral periodontal flora imbalance can be monitored early.

In addition, the marker set provided by the invention can be used as one of the projects of regular dental examination or part of large-scale screening to regularly and dynamically monitor the early diagnosis and the periodontitis development state of the subjects with a wider population, and is beneficial to the 'early discovery and early treatment' of the periodontitis.

Further, the marker panel provided by the present invention is also suitable for the assessment of the effect of a treatment regimen on a patient. For patients diagnosed with periodontitis and treated over time, the values of each marker set can be used to examine whether periodontitis has improved (e.g., a decrease in the value of one or more markers over time) and whether the treatment regimen needs to be adjusted (e.g., if the value of one or more markers does not increase or increase significantly over time, indicating that the treatment regimen may not be appropriate for the subject), thus facilitating the implementation of personalized treatment.

In particular, the marker set provided by the invention is suitable for dynamic monitoring of periodontitis, and for example, an authorized oral doctor and a subject can obtain historical data of each marker obtained in each detection on a terminal.

Example one

This example included 18 patients with mild periodontitis, 16 patients with severe periodontitis and 22 people with periodontal health. Wherein, the clinical diagnosis standard of periodontitis is as follows: the adjacent surface adhesion loss (CAL) exists in 2 or more than 2 non-adjacent teeth, or the buccal (lingual) surface with more than or equal to 2 teeth has the adhesion loss of more than or equal to 3mm, and the periodontal pocket with more than 3mm is provided. The clinical diagnostic criteria for mild periodontitis (including mild and moderate periodontitis) are: inflammation and probing Bleeding (BOP) exist in gingiva, periodontal pocket is less than or equal to 4mm, attachment loss is 1-2mm, and X-ray film shows that alveolar bone resorption does not exceed 1/3 of root length. The clinical diagnosis criteria for severe periodontitis are: periodontal pocket is more than 6mm, attachment loss is more than or equal to 5mm, X-ray film shows that alveolar bone absorption exceeds 1/2 of root length, multiple teeth have root bifurcation lesion, teeth are mostly loose, inflammation is obvious or periodontal abscess can be generated. The clinical diagnosis standard of healthy periodontal is: there is no or very little clinical inflammation in normal periodontal tissue, the depth of periodontal probing is no more than 3mm, and the site of probing bleeding is < 10%. All subjects had no serious systemic and immune system disease.

The invention finds that the accurate distinction of periodontal healthy people and patients with mild periodontitis can be realized by combining the five-dimensional markers of the quantity of gingival crevicular fluid, the pH of the gingival crevicular fluid, gingival crevicular fluid hemoglobin, a porphyromonas gingivalis-related marker and a periodontitis-related biochemical marker, as shown in Table 1.

TABLE 1 differentiation of periodontal healthy population from patients with mild periodontitis

The accuracy related to the invention is calculated by the following formula:

wherein TP represents True Positive (True Positive), TN represents True Negative (True Negative), FP represents False Positive (False Positive), and FN represents False Negative (False Negative).

On the whole, the combination of the five-dimensional markers is used as a marker group for detecting periodontitis, and the differentiation between periodontal healthy people and patients with mild periodontitis can be achieved more specifically, comprehensively, accurately and effectively. This provides assistance for the early diagnosis of periodontitis, and even enables the diagnosis of periodontitis earlier than the current clinical examination.

The above data also show that for less economically developed areas or where relatively low accuracy requirements are required (e.g., large scale screening), marker sets of the three dimensions of the amount of gingival crevicular fluid, the pH of the gingival crevicular fluid, the gingival crevicular fluid hemoglobin and the periodontitis-related bacteria can be selectively employed, and while a certain accuracy is ensured, the number of markers detected can be reduced to reduce the cost of the detection scheme and promote the popularization of periodontitis detection.

Example two

Next, this example further verifies whether the marker set can accurately identify patients with mild periodontitis and patients with severe periodontitis, and the results are shown in table 2. The result shows that the marker group provided by the invention not only can realize the differentiation of healthy periodontal crowds and patients with mild periodontitis, but also can realize the accurate diagnosis of patients with mild periodontitis and patients with severe periodontitis. In conclusion, the marker set provided by the invention can accurately identify and distinguish the development stage of periodontitis and can be used as an effective basis for detecting periodontitis.

TABLE 2

	Accuracy of marker set provided by the invention
		Severe periodontitis patients	97.2％
Patients with mild periodontitis	87.5％

In clinical examination, the early development stage of periodontitis and the boundary of periodontal health are not very obvious, which not only brings trouble to oral doctors, but also hinders the 'early discovery and early treatment' of periodontitis. As shown in table 3, the marker set provided by the present invention not only can accurately identify and distinguish the progression stages of periodontitis (zeroth stage-periodontal health, first stage-mild periodontitis, and second stage-severe periodontitis), but also can determine the progression tendency of periodontitis (e.g., the progression or improvement of periodontitis) of a subject according to the values of the markers in the marker set measured by the subject each time. This not only provides early warning of the progression of periodontitis, but also provides a new angle of assessment of the treatment regimen, e.g., as judged by the value of each marker, whether the subject's stage of periodontitis development shifts from the first stage or second stage to the zeroth stage to assess whether the subject's periodontitis is relieved, controlled, improved, or restored to a healthy state by the received periodontal treatment.

The marker set provided by the invention can evaluate the periodontitis state of a subject from multiple dimensions, and the determination result is easy to visualize (for example, the form of the determination result can be further processed and more intuitively reflected), so that the individual treatment can be better realized by an oral doctor according to the actual determination result of the subject by 'pertinently giving medicines', and the accurate evaluation of a treatment scheme and the maintenance condition after the treatment by the oral doctor and/or a periodontitis patient can be facilitated.

TABLE 3

EXAMPLE III

In order to facilitate early evaluation of changes in the progression state of periodontitis in a subject, or to quickly screen out subjects who may have periodontitis or who are at risk of worsening periodontitis among a large number of human subjects in oral cavity examination activities of a large population, the present invention provides an extraction device for extracting a gingival crevicular fluid sample, as shown in fig. 1 to 5, the extraction device comprising:

an extraction tube 10, said extraction tube 10 comprising an extraction end 12, and an outlet end disposed distal to said extraction end, wherein said extraction end 12 is sized and shaped to extend between a tooth and an adjacent gum, wherein a side of said extraction tube 10 proximal to said extraction end 12 is configured as a capillary channel 11, and wherein said extraction tube is provided with at least one air pressure balance tube 13;

a mixing chamber 20, wherein the mixing chamber 20 comprises a containing space 21 for containing the sample, the containing space 21 is communicated with the outlet end, and the containing space is provided with a first opening at the position corresponding to the outlet end;

a pushing part 30, a first end 31 of which can extend into the interior of the extraction tube 10 from the outlet end, and a second end 32 of which penetrates through the accommodating space 21 and extends outwards from a first opening arranged on the accommodating space 21; when the pushing part 30 moves along a first direction, the sample in the capillary 11 can be driven to flow along the first direction by the action of the pushing part on the air inside the extraction tube, wherein the first direction is from the extraction end to the outlet end.

In some embodiments, the extraction tubes 10 are all provided as elongated capillary channels.

In some embodiments, the side of the extraction end of the extraction tube 10 is configured as a capillary channel, while the side thereof near the outlet end may be configured as a generally sized channel (i.e., a non-capillary channel).

In embodiments of the invention, the extraction tube is capable of spontaneous collection of gingival crevicular fluid by capillary action, and thus the amount of extracted gingival crevicular fluid obtained is correlated to some extent with the degree of periodontitis experienced by the subject. For example, a subject with severe periodontitis may extract more gingival crevicular fluid at the same amount of capillary action and the same extraction time.

In some embodiments, the extraction tube 10 is provided with at least one graduated line 14 that can be used to read the amount of the sample extracted.

In some embodiments, the capillary channel has an inner diameter of about 0.4mm to about 0.6mm.

In some embodiments, two sections of scale marks are sequentially arranged on the capillary tube at intervals, and the gingival crevicular fluid content readings corresponding to the two sections of scale marks are as follows: 10. Mu.L and 20. Mu.L.

Or, in some embodiments, four segments of scale marks are sequentially arranged on the capillary tube at intervals, and the reading of the gingival crevicular fluid content corresponding to the four segments of scale marks is respectively: 10. Mu.L, 20. Mu.L, 40. Mu.L, 50. Mu.L.

For example, in some embodiments, the inner diameter of the capillary channel is about 0.4mm, and the length of the corresponding capillary channel is set to about 10cm to 12cm. And the positions of about 2cm, 4cm, 8cm and 10cm away from the extraction end on the capillary pipeline are respectively provided with a section of scale mark. Wherein, when the liquid level of the gingival crevicular fluid is positioned between the graduation lines of 2cm-4cm, the content of the gingival crevicular fluid is about 10-20 μ L; when the liquid level of the gingival crevicular fluid is between the graduation lines of 4cm-8cm, the gingival crevicular fluid content is about 21-40 muL; when the level of the gingival crevicular fluid is between 8-10cm, it represents a gingival crevicular fluid content of between about 41-50 μ L. In the embodiment, the content interval of the gingival crevicular fluid can be quickly read by means of the scale marks.

As another example, in some embodiments, the capillary channel has an inner diameter of about 0.5mm and a length correspondingly set at about 7-10cm. And the positions of about 1.3cm, 2.5cm, 5.1cm and 6.4cm away from the extraction end on the capillary pipeline are respectively provided with a section of scale mark. Wherein, when the liquid level of the gingival crevicular fluid is positioned between the graduation lines of 1.3cm-2.5cm, the content of the gingival crevicular fluid is about 10-20 μ L; when the liquid level of the gingival crevicular fluid is between the graduation lines of 2.5cm-5.1cm, the gingival crevicular fluid content is about 21-40 muL; when the level of the gingival crevicular fluid is between 5.1 and 6.4cm, it represents a gingival crevicular fluid content of between about 41 and 50 μ L.

For another example, in other embodiments, the capillary channel inner diameter is about 0.6cm, and its length is correspondingly set to about 4.5-6cm. Correspondingly, a section of scale mark is respectively arranged on the capillary pipeline along the positions 0.9cm, 1.8cm, 3.5cm and 4.4cm away from the extraction end.

In the embodiment of the invention, the spontaneous collection function of the extraction tube and the arrangement of the scale marks are convenient for a user to accurately and quickly quantitatively read the extraction amount of the gingival crevicular fluid. It will be appreciated that the reading of the extracted amount of gingival crevicular fluid during use may be recorded as a specific numerical value (and correspondingly, more graduations may be provided on the extraction tube to facilitate more accurate readings) or as a range of values.

In some embodiments, the extraction tip is blunt to avoid or reduce damage to the gums during extraction.

In some embodiments, the first end 31 of the pushing portion has an outer diameter dimension greater than an outer diameter dimension of the second end 32, and correspondingly, the outlet end of the extraction tube has an inner diameter dimension greater than an inner diameter dimension of the first opening.

For example, in some embodiments, as shown in fig. 2 and 3, the outlet end of the extraction tube 10 extends into the accommodating space 21, and the accommodating space 21 is further provided with a first opening for the pushing part 30 to pass through (specifically, the first opening may be a tubular structure formed by extending the accommodating space outwards, and the first opening is located on an extension line of the central line of the extraction tube). Wherein, the inner diameter size of the outlet end is slightly larger than the inner diameter size of the first opening, and the first end 31 of the pushing part is larger than the inner diameter of the second end 32 thereof, so that the first end of the pushing part can move back and forth in the extraction tube 10, but the first end of the pushing part can not pass through the first opening, that is, the pushing part can not be pulled out completely, thereby limiting the moving distance of the pushing part to a certain extent.

For another example, in some embodiments, a side of the pushing portion close to the first end is provided with a limiting ring, and an outer diameter of the limiting ring is larger than an inner diameter of the first opening, so as to limit a moving distance of the pushing portion and prevent the pushing portion from being completely pulled out.

In some embodiments, the pushing portion is made of a rubber material, and an outer diameter dimension of the pushing portion in a natural state (i.e., a state in which no external force is applied) is larger than an inner diameter dimension of the first opening. Therefore, when the pushing portion made of the rubber material is in mutual contact with the outlet end and the first opening, the rubber material is extruded and deformed to a certain degree under the extrusion effect of the first opening and the outlet end, so that the rubber material is in close contact with the first opening and the outlet end, and a good sealing effect is achieved. And the rubber material is closely contacted with the outlet end and the first opening, so that the pushing part can be directly and completely pulled out (equivalently, a first resistance for limiting the movement of the pushing part is provided for the pushing part) due to overlarge external acting force in the using process to a certain extent.

In some embodiments, the extraction tube is provided as a frangible structure proximate a side of the mixing chamber. After extraction is completed using the extraction device, the length of tubing between the break-off structure and the extraction end can be broken off to provide the mixing chamber 20 as the final sample storage unit.

Alternatively, in other embodiments, the extraction tube 10 comprises a stationary tube and a movable tube detachably connected to each other.

Specifically, in some embodiments, the fixed pipe is fixedly connected with the mixing chamber, and one end of the fixed pipe extending into the accommodating space is an outlet end, and the other end of the fixed pipe is detachably connected with one end of the movable pipe. Wherein, the other end of the movable tube is the extraction end, and the movable tube is set to be a capillary structure. In this embodiment, the movable tube that directly contacts the oral cavity of the subject is used as a disposable, and the other parts of the extraction device can be reused after cleaning and disinfection, so that the detection cost of periodontitis can be reduced to some extent.

Further, in some embodiments, the stationary tube is made of a rubber material (or, in other embodiments, the portion of the stationary tube that mates with the movable tube is made of a rubber material), and the movable tube is made of plastic, and the outer diameter dimension of the movable tube may be slightly larger than the inner diameter dimension of the rubber material. Wherein the movable pipe can be connected with the fixed pipe by being inserted into the inner wall of the fixed pipe. In this embodiment, can realize closely cooperating (avoiding liquid excessive) between fixed pipe and the movable tube, dismantle the change between the two simultaneously is simple, easy operation.

In some embodiments, the outlet end of the extraction end may also be used as a liquid outlet from the receiving space.

Alternatively, in other embodiments, the accommodating space is provided with a liquid outlet, and the liquid outlet is provided with a sealing opening. In the process of storage and transportation of the extraction device, the sealing opening can ensure the cleanness of the inner space. The sealed opening can be opened when the sample in the receiving space needs to be taken out.

In some embodiments, the mixing chamber 20 is pre-filled with a diluent. Suitable diluents are, for example, TES, MES, PIPES, HEPES, MOPS, tris, PBS and physiological saline.

In some embodiments, the air pressure balancing tube 13 is provided with a sealed opening, and when the extraction device is used to extract gingival crevicular fluid, the air pressure balancing tube 13 is required to balance the air pressure inside and outside the capillary channel, and the sealed opening is opened. When it is necessary to pull the push portion 30 so that the liquid inside the capillary tube can enter the containing space 21, the seal opening may be closed to limit the action of the air pressure equalizing tube 13 to equalize the air pressure.

Alternatively, in other embodiments, the gas pressure equalization tube 13 is made of a bendable material (e.g., plastic) wherein the gas pressure equalization tube extends a length outwardly from the main portion of the extraction tube. When the effect of the balanced air pressure of the air pressure balance tube needs to be limited, the bent air pressure balance tube can be pressed (i.e. the air pressure balance tube achieves a closed effect and cannot help air exchange or flow between the interior of the extraction tube and the external environment), so that the liquid sample in the capillary tube can smoothly enter the accommodating space 21 under the effect of the pushing part.

In some embodiments, the extraction device is also provided with a label (e.g., on the outer surface of the mixing chamber). After the user reads out the extraction volume of gingival crevicular fluid, can record the extraction volume reading on the label, the record management of the data of being convenient for.

To further explain the technical solution in the embodiment of the present invention, the following describes the use process of the above-mentioned extraction apparatus with reference to fig. 5:

when the extraction device is not activated, the first end of the pushing part is positioned inside the outlet end of the extraction tube to seal the diluent pre-stored inside the containing space (prevent the diluent from entering the extraction tube).

In use, the extraction end of the extraction device is extended between the tooth and the adjacent gum, as shown in figure 5, so that gingival crevicular fluid is wicked into the interior of the capillary channel. After extraction is finished, the extraction amount of gingival crevicular fluid is read through the scale marks, and corresponding data are recorded. Then the gingival crevicular fluid extraction device is inverted (or transversely placed), and the pushing part is pulled along the first direction, so that the liquid in the capillary tube enters the mixing cavity under the dual actions of gravity and the pushing part and is mixed with the pre-stored diluent.

Finally, the lower half of the extraction tube 10 (i.e., the side close to the extraction end) is broken off from the breaking structure 15, and the pushing portion 30 is pushed back to the outlet end of the extraction tube 10 again, so that the mixed solution of the gingival crevicular fluid and the diluent inside the mixing cavity 20 is hermetically preserved.

In this embodiment, the user (e.g., the inspector) can conveniently and quickly extract the gingival crevicular fluid and mix the gingival crevicular fluid with the diluent. Wherein, the mixed storage of gingival crevicular fluid and diluent can reduce the loss of gingival crevicular fluid to a certain extent to the save time of extension gingival crevicular fluid. Moreover, the mixed preservation mode of the gingival crevicular fluid and the diluent adopted in the embodiment can reduce the loss of the gingival crevicular fluid in the processes of transportation, storage and the like, and conversely can reduce the necessary extraction amount of the gingival crevicular fluid to a certain extent (namely, the gingival crevicular fluid amount capable of meeting the detection requirement). Therefore, the extraction operation times or the extraction operation time of the gingival crevicular fluid can be reduced in the actual extraction process, so that the damage of the extraction device to the gingiva of a subject (such as a periodontitis patient) can be reduced or avoided to a certain extent, and the extraction efficiency of the gingival crevicular fluid can be improved to a certain extent.

Meanwhile, the mixing cavity 20 with a more regular structure and a smaller occupied space is used as an independent storage unit subsequently, so that the extraction device (or gingival crevicular fluid extracted in batches) after being used in batches can be stored and transported integrally.

Under the synergistic effect of multiple effects of smaller gum damage, higher extraction efficiency, convenience for batch storage and the like, the gingival crevicular fluid extraction device disclosed by the invention can be well suitable for an application scene of rapidly screening oral health conditions (such as whether periodontitis exists) of large-scale people.

For example, the extraction device of the present invention can be widely applied to oral cavity diagnostic activities performed by organizations such as communities for local residents, or activities for performing free oral cavity health detection on teachers and students when healthy oral cavities enter campuses. In these large-scale screening activities, the number of subjects is large, and the sampling and detection work is very heavy and the efficiency requirement is very high. And this mass screening activity has been associated with the problem of long-term on-site sampling, off-site testing, etc. (e.g., requiring a resident to be sampled on-site for a long period of time in an area remote from a hospital or testing laboratory, and then transporting the sample to the hospital or testing laboratory). The extraction device provided by the invention can reduce the necessary extraction amount of gingival crevicular fluid on one hand, so that the sampling efficiency is greatly improved when a single subject is sampled, and the efficient sampling can effectively reduce the gingival damage to the subject. After the sample is ended, the user also can record the extraction volume reading of gingival crevicular fluid fast to "the hybrid of gingival crevicular fluid, diluent is deposited fast.

The batch extraction device for a plurality of subjects can be stored and transported in batch in a minimized unit form (namely a mixing chamber), so that the sampled products can be conveniently transported from a sampling site of a community, a campus and the like to a hospital or a detection laboratory at a different place to complete subsequent detection. In addition, in the process of storage and transportation, the mixed storage can reduce the loss of gingival crevicular fluid so as to prolong the effective preservation time of the gingival crevicular fluid.

Preferably, such large-scale batch testing programs can be tested by a) the amount of gingival crevicular fluid; b) pH of gingival crevicular fluid; c) The periodontal healthy population and the periodontitis patient can be rapidly distinguished by the markers with three dimensions, such as gingival crevicular hemoglobin, so that the 'early discovery and early treatment' of periodontitis can be realized with lower economic cost.

Example four

As shown in fig. 6-9, the present embodiment provides another extraction device for extracting a gingival crevicular fluid sample, the extraction device comprising:

a first storage chamber 40, wherein a first storage space 42 for containing the sample is formed in the first storage chamber 40, the first storage chamber 40 further comprises a pushing part 30, the first end 31 of the pushing part 30 is positioned in the first storage chamber 40, and the second end 32 of the pushing part 30 extends out of the first storage chamber 40;

an extraction tube 10, wherein the extraction tube 10 comprises an extraction end 12 and an outlet end arranged at a side far away from the extraction end 12, wherein the outlet end is communicated with the first storage space 42, the extraction end 12 is sized and shaped to extend between a tooth and an adjacent gum, a capillary channel 11 is arranged at a side of the extraction tube 10 close to the extraction end 12, and the outlet end of the extraction tube 10 is communicated with the second storage space;

a second storage cavity 50, a second storage space 52 for containing liquid is formed in the second storage cavity 50, and the second storage space is communicated with the first storage space through a first channel, wherein at least one air pressure balancing port is further arranged in the second storage space 52, a first channel 53 extends outwards from the second storage cavity 50, the first channel 53 is communicated with the second storage space 52, and one side of the first channel 53, which is close to the second storage space, is provided with a capillary structure;

when the pushing part 30 moves in a direction away from the first storage chamber 40, the sample in the capillary channel and the liquid in the second storage chamber 50 can be driven to flow towards the first storage chamber 40 by the action of the pushing part 30 on the air in the extraction device.

It should be noted that different terms, such as "receiving space", "first storage space" or "second storage space", are used in embodiments of the present invention to describe a chamber for storing a liquid sample (e.g., gingival crevicular fluid or a diluent), such as a mixing chamber, or a storage chamber, etc., primarily to facilitate distinguishing between different chambers in different embodiments. In fact, in the embodiment of the present invention, the above various accommodating spaces or storage spaces may have the same structure. Of course, different configurations are possible. These chambers may be provided with structures including: spherical cavities, or rectangular cavities, etc.

In some embodiments, the extraction tube 10 includes a fixed tube fixedly connected to the second storage space 52, and a movable tube having one end detachably connected to the fixed tube, and the other end of the movable tube is the extraction end.

In this embodiment, the movable tube portion is a disposable portion, and the remainder of the extraction device may be reused after corresponding cleaning operations such as sterilization. The cost of periodontitis detection is reduced to a certain extent.

For example, in some embodiments, the stationary pipe is made of a rubber material (or, in other embodiments, a portion of the stationary pipe, which is engaged with the movable pipe, is made of a rubber material), and the movable pipe is made of a plastic material, wherein the movable pipe is inserted into an inner wall of the stationary pipe to be coupled with the stationary pipe. In this embodiment, can realize closely cooperating (avoiding liquid excessive) between fixed pipe and the movable tube, dismantle the change between the two simultaneously is simple, easy operation.

In some embodiments, the first storage space 40 is provided with a second opening 41.

In this embodiment, the second opening 41 may function to equalize the air pressure. When the extraction tube is used to extract gingival crevicular fluid from the gingiva of the subject, the second opening is made to be in an open state, which can be used to equalize the air pressure inside the extraction device.

Further, in some embodiments, the second opening may also function as a fluid outlet for draining mixed gingival crevicular fluid and diluent fluid inside the second storage chamber.

In some embodiments, a sealed opening is disposed over the second opening. The sealing opening can seal the second opening to ensure the cleanness of the inner space during the storage and transportation of the extracting device. The sealed opening can be opened when the second opening is required to function as a drain or to equalize air pressure.

In some embodiments, the extraction tube has at least one graduated line disposed thereon that can be used to read the amount of extraction of the sample.

In some embodiments, the extraction end is rounded.

In some embodiments, the first end of the push portion has a size greater than a size of the second end.

In some embodiments, the pushing portion is made of a rubber material.

In some embodiments, the first channel is a capillary channel for connecting the first and second storage spaces, and the capillary channel can restrict the flow of the diluent in the second storage space.

Specifically, a first end of the capillary channel is connected to the first storage space, and a second end thereof is connected to the second storage space. At this time, since the inner diameter of the first end of the capillary channel is much smaller than the size of the second storage space, a part of the diluent in the second storage space may enter the first end of the capillary channel under the capillary action (it can be understood that, since the second storage space is in a sealed state, the content of the diluent entering the inside of the capillary channel under the air pressure is not excessive). Moreover, since the size of the first storage space (specifically, the inner diameter of the first storage space at the connection with the capillary channel) is larger than the inner diameter of the second end of the capillary channel, the liquid entering the capillary channel is difficult to continue to enter the first storage space through the second end (or the size of the first storage space is larger than the size of the second end of the capillary channel, which is equivalent to a second resistance for the diluent to flow into the first storage space). Therefore, the first channel in the embodiment has a flow limiting function, so that the diluent is further prevented from entering the extraction pipe and polluting the extraction pipe.

Of course, in other embodiments, the first channel may be formed by a combination of a common size tube and a capillary tube. Specifically, a side of the first channel adjacent to the second storage space is provided as a capillary channel.

In some embodiments, to avoid the elongate first channel from being broken, a protective sleeve is further provided outside the first channel.

It can be understood that, unlike the third embodiment of the present invention, in the present embodiment, a relatively independent storage space is designed for the diluent, and a corresponding flow-limiting structure (i.e., the first channel) is adopted to assist in achieving stable storage of the diluent. Therefore, the storage amount of the diluent can be increased to a certain extent, and the diluent cannot be immersed into the extraction tube to pollute the extraction tube due to excessive diluent. Therefore, compared with the extraction device in the third embodiment, the extraction device in this embodiment can provide more test samples (i.e., a mixed solution of gingival crevicular fluid and diluent) to some extent, thereby satisfying a relatively more test scenario for test items.

For example, the propensity of an oral patient to develop periodontitis or the severity of periodontitis experienced may be relatively high for an oral patient who visits or visits the hospital autonomously. Therefore, for such patients with self-diagnosis, providing sufficient detection samples facilitates the selection of more comprehensive and accurate detection schemes. Specifically, the patient who makes a diagnosis by himself can comprehensively detect the quantity of gingival crevicular fluid, the pH of the gingival crevicular fluid, gingival crevicular fluid hemoglobin, the quantity of porphyromonas gingivalis, and multiple indexes of gingival proteinase K, matrix metalloproteinase-8, interleukin-6, interleukin-1 beta, 8-hydroxydeoxyguanosine, malondialdehyde and the like.

It should be understood that the above is only a description of a preferred application scenario of the embodiment of the present invention, and actually, the extraction device in either the third embodiment or the fourth embodiment can be applied to different screening activities, such as large-scale community detection or hospital field detection.

The technical scheme in the embodiment of the invention is further explained by combining the use process of the extraction device as follows:

as shown in fig. 9, the extraction tip of the extraction device extends into the gum of the subject to extract gingival crevicular fluid, and the second opening is opened to allow the second opening to act as a pressure equalization valve.

After extraction of gingival crevicular fluid is completed, closing the second opening, inverting the extraction device, and pulling the pushing portion outward in a direction away from the first storage space so that the diluent in the second storage space enters the first storage space under the action of gravity and the pushing portion (in the process, the second resistance is overcome); likewise, gingival crevicular fluid in the capillary channel enters the first storage space under the action of gravity and the push portion. Subsequently, the gingival crevicular fluid and the diluent are mixed with each other in the first storage space. Finally, the mixed liquid can be discharged by utilizing the second opening so as to complete the corresponding detection item.

EXAMPLE five

As shown in fig. 10a to 10c, this embodiment further provides a detection card matched with any one of the extraction devices in the third and fourth embodiments, and the detection card includes:

a body 60;

the sample loading port 61 is arranged on the body, and the opening of the sample loading port is arranged on the first surface of the body;

a plurality of first detection regions 62, and the first detection regions 62 are in communication with the sample injection port 61 through a second channel 63, wherein a first end of the second channel is in communication with the sample injection port 61, and a second end of the second channel is in communication with the first detection regions 62.

In some embodiments, a plurality of the first detection regions 62 may be provided. For example, 4 to 9 may be generally set.

In some embodiments, the first detection zone may be pre-deposited with a reagent that can be used to detect the value of a label or a compound related to the value of a label, wherein the reagent may be an aptamer corresponding to the label (corresponding to the target). For example, in a specific embodiment, 9 first detection regions are disposed on the detection card, wherein 1 first detection region serves as a blank control, and the other 8 first detection regions serve as a test group, and the 8 first detection regions are respectively provided with an aptamer capable of specifically recognizing human hemoglobin, an aptamer capable of specifically recognizing porphyromonas gingivalis, an aptamer capable of specifically recognizing matrix metalloproteinase-8, an aptamer capable of specifically recognizing interleukin-6, an aptamer capable of specifically recognizing interleukin-1 β, an aptamer capable of specifically recognizing 8-hydroxydeoxyguanosine, an aptamer capable of specifically recognizing malondialdehyde, and an aptamer capable of specifically recognizing gingivasin K.

Of course, any one or more of the above reagents may be pre-stored on the detection card according to the current actual detection requirements (e.g., detection accuracy).

In some embodiments, the aptamer is a single-stranded DNA molecule.

In some embodiments, the aptamer is modified with a fluorophore and/or a quencher. For example, fluorescent labeling techniques can covalently attach a fluorophore to an aptamer. The technology uses the fluorescence characteristic of the fluorescent group to provide the signal of the detected object by reacting the specific group of the fluorescent signal substance with the specific group of the aptamer so as to complete the labeling process. Among them, rhodamine and fluorescein are the most commonly used reagents for providing fluorescent genes, and other fluorescent labeling reagents, such as polycyclic aromatic hydrocarbon compounds, heteroaromatic compounds, and chelates of some rare earth elements, can also be used.

In some embodiments, the aptamer may also be prepared into an aptamer sensor by using a nanomaterial as a carrier, wherein the nanomaterial is often selected from the following: metal and metal oxide nanoparticles include gold nanoparticles, silver nanoparticles (AgNPs), platinum nanoparticles (PtNPs), iron oxide nanoparticles (iopns), molybdenum nanoparticles, and the like.

In some embodiments, the detection card is further provided with a second detection zone for detecting the PH of the mixed solution. For example, the second detection area is a rectangular groove disposed on the surface of the detection card, and a PH test paper may be disposed in the rectangular groove for detecting the PH value of the mixed solution. In this embodiment, a user (e.g., a tester) may read the PH value by manually comparing with the standard color chart.

In some embodiments, the second channel 63 may be disposed inside the test card.

Alternatively, in other embodiments, a second channel (i.e., a sample dispensing channel) may be provided on the second surface of the test card and sealed by covering the second surface with a sealing film.

In some embodiments, the second end of the second channel 63 is configured as a capillary channel, and the inner diameter of the capillary channel is configured to gradually decrease in a direction approaching the first detection zone 62.

In this embodiment, when the reagent in the liquid state is stored in the first detection area 62, the inner diameter of the second channel 63 is gradually changed to limit the reagent in the first detection area 62 from overflowing into the second channel 63. Alternatively, even if some of the reagent overflows into the second end of the second channel 63, the overflow reagent in the second end can be prevented from further moving towards the first end of the second channel 63, even into the sample port 61, and contaminating the card (it will be understood that the liquid will not actively flow from the place with the larger capillary action to the place with the smaller capillary action without the action of other external forces). Conversely, when the mixed liquid in the sample addition port enters the first detection area 62 along the second channel 63, the second channel can also guide and flow the mixed liquid, so that adverse effects on the sample separation process of the mixed liquid are avoided.

In some embodiments, the second channel may be configured to have some capillary action.

For example, in one embodiment, in use, a mixed solution is added to the sample addition port via the liquid outlet of the gingival crevicular fluid extraction device, and the mixed solution can be wicked (or, alternatively, can be combined with centrifugal force) through the second channel into each of the first detection zones. The test card is then inserted into the test instrument in a horizontal configuration (and in other embodiments, in a vertical configuration), and the test instrument can read the data (e.g., read the color of the test fields) from each of the first test fields, wherein the use of the test card with the test instrument 70 is illustrated in fig. 11-13.

Of course, it will be appreciated that the test card may also be adapted to interact with the test instrument in other forms than that shown in FIGS. 11-13.

Specifically, in some embodiments, the detection instrument includes a housing, a detection card placing cavity for placing a detection card is provided inside the housing, and a first detection assembly, the first detection assembly including: the detection card placing cavity is provided with a first image acquisition module (such as a fluorescence microscope or other fluorescence cameras for acquiring fluorescence pictures of a first detection area) and a first image analysis module, wherein the first image acquisition module is arranged corresponding to the detection card placing cavity, and the first image analysis module is connected with the first image acquisition module. Wherein, after the user placed the detection card in the detection card places the chamber to start detecting instrument, detecting instrument's main work flow is as follows: 1) The fluorescence microscope respectively collects the fluorescence images of the first detection areas and sends the collected images to the first image analysis module; 2) The first Image analysis module (for example, image J Image analysis software may be used) acquires an Image, splits the Image into 3 channels of red, green and blue, and then performs fluorescence signal identification on the green channel map to obtain an average fluorescence intensity.

Wherein the average fluorescence intensity (corresponding to the value of the marker) in each first detection zone can be compared with the fluorescence intensity information (corresponding to the reference value of the "marker") of different standard samples. For example, in the case of hemoglobin detection, before the detection, a plurality of sets of hemoglobin standard samples with different concentrations can be prepared in advance, and fluorescence intensity information (e.g., average fluorescence intensity) of the hemoglobin standard samples with different concentrations after reacting with the same reagent (e.g., aptamer specifically recognizing human hemoglobin) under the same reaction environment can be tested and recorded. Finally, the fluorescence intensity of the test sample is compared with the fluorescence intensity of the standard sample by a detection person or corresponding computer software, so as to obtain a corresponding judgment result (for example, the hemoglobin concentration of the sample).

For another example, in some embodiments, the detection instrument further comprises: a second detection assembly, wherein the second detection assembly comprises: the second image acquisition module is arranged towards a second detection area on the detection card, and the second image analysis module is connected with the second image acquisition module. Specifically, the second image acquisition module is a video camera (or also a camera), and the second image analysis module is a single chip microcomputer (or also other electronic chips). After the first detection assembly finishes the detection work, the second detection assembly starts to operate, wherein the camera shoots the image of the second detection area and sends the image to the single chip microcomputer, and the single chip microcomputer measures the pH value of the sample to be detected in the second detection area through the contrast color block.

Preferably, the second detection assembly may further include: an illumination module, likewise arranged towards the second detection area, can provide a suitable light source when the camera takes an image.

Therefore, under the mutual cooperation of the detection card and the detection instrument, the collected gingival crevicular fluid can be subjected to detection reactions with different dimensions at one time, and the values of various markers or the comparison result of the values of the markers and the reference value can be synchronously output by the detection instrument, so that the periodontitis development state of the subject can be conveniently and rapidly evaluated.

EXAMPLE six

The embodiment provides a periodontitis detection kit, and the kit comprises the extraction device in the third embodiment or the fourth embodiment.

Further, in some embodiments, the periodontitis detection kit further comprises: any one of the test cards of example five. While the present invention has been described with reference to the embodiments shown in the drawings, the present invention is not limited to the embodiments, which are illustrative and not restrictive, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Use of a marker panel in the manufacture of a kit for assessing the periodontitis state of a subject, the marker panel comprising:

a) Amount of gingival crevicular fluid;

b) pH of gingival crevicular fluid;

c) Gingival crevicular hemoglobin;

2. The use of claim 1, wherein the set of markers further comprises a Porphyromonas gingivalis-associated marker comprising an amount of Porphyromonas gingivalis and Gum proteinase K.

3. The use of claim 1, wherein the set of markers further comprises biochemical markers associated with periodontitis including matrix metalloproteinase-8, interleukin-6, interleukin-1 β, 8-hydroxydeoxyguanosine, and malondialdehyde.

4. The use according to claim 1, wherein the periodontitis trend comprises a worsening of periodontitis or a amelioration of periodontitis.

5. The use of claim 4, wherein the subject is assessed as having a strong positive trend for the progression of periodontitis when all of the markers in the marker set are above a reference value; (ii) the subject is assessed as having a positive trend for progression of the periodontitis when one or more markers in the marker set are above the reference value; (ii) the subject is assessed as having a negative trend of progression of the periodontitis when none of the markers in the marker set is above the reference value; the subject is assessed as having a trend towards an improvement in the periodontitis when none of the markers in the marker set are above the reference value and one or more of the markers are below the reference value.

6. The use of claim 1, wherein the stage of periodontitis progression comprises a periodontal health stage, a mild periodontitis stage, and a severe periodontitis stage.

7. The use of claim 1, wherein the subject comprises a periodontitis patient.

8. A marker panel for assessing a subject's periodontitis status, including stage of periodontitis progression and/or periodontitis progression trend, the marker panel comprising:

a) Amount of gingival crevicular fluid;

b) pH of gingival crevicular fluid;

c) Gingival crevicular fluid hemoglobin.

9. The marker panel of claim 8 further comprising a Porphyromonas gingivalis-related marker comprising an amount of Porphyromonas gingivalis and Gum proteinase K.

10. The marker panel of claim 8, wherein the marker panel further comprises biochemical markers associated with periodontitis including matrix metalloproteinase-8, interleukin-6, interleukin-1 β, 8-hydroxydeoxyguanosine, and malondialdehyde.