Detection kit and detection method for periodontal pathogen infection marker
阅读说明：本技术 一种牙周致病菌感染标记物检测试剂盒和检测方法 (Detection kit and detection method for periodontal pathogen infection marker ) 是由 农高惠 何林声 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种牙周致病菌感染标记物检测试剂盒,包括样本洗脱液、髓过氧化物酶检测试剂和牙龈蛋白酶检测试剂,样本洗脱液为三羟甲基氨基甲烷、3-吗啉丙磺酸、4-羟乙基哌嗪乙磺酸、哌嗪-N,N’-二(2-乙磺酸)或2-(N-吗啡啉)乙磺酸水溶液；髓过氧化物酶检测试剂包括氧化酶、氧化酶对应的底物和显色剂A,氧化酶和其对应的底物相遇后能够产生过氧化氢,显色剂A包括Trinder反应色原底物；牙龈蛋白酶检测试剂为牙龈蛋白酶检测试剂I或II,牙龈蛋白酶检测试剂I包括底物BAEE、乙醇氧化酶和显色剂B,显色剂B包括过氧化物酶和Trinder反应色原底物；牙龈蛋白酶检测试剂II包括BANA和重氮盐显色剂。(The invention discloses a detection kit for periodontal pathogen infection markers, which comprises a sample eluent, a myeloperoxidase detection reagent and a gingipain detection reagent, wherein the sample eluent is aqueous solution of tris (hydroxymethyl) aminomethane, 3-morpholine propanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, piperazine-N, N' -di (2-ethanesulfonic acid) or 2- (N-morpholine) ethanesulfonic acid; the myeloperoxidase detection reagent comprises oxidase, a substrate corresponding to the oxidase and a color-developing agent A, wherein the oxidase can generate hydrogen peroxide after meeting with the substrate corresponding to the oxidase, and the color-developing agent A comprises a Trinder reaction chromogen substrate; the gingival protease detection reagent is a gingival protease detection reagent I or II, the gingival protease detection reagent I comprises a substrate BAEE, an alcohol oxidase and a color-developing agent B, and the color-developing agent B comprises a peroxidase and a Trinder reaction chromogen substrate; the gingival protease detection reagent II comprises BANA and a diazonium salt color developing agent.)
1. A periodontal pathogen infection marker detection kit is characterized in that: comprises a sample eluent, a myeloperoxidase detection reagent and a gingipain detection reagent,
the sample eluent is aqueous solution of tris, 3-morpholine propanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, piperazine-N, N' -di (2-ethanesulfonic acid) or 2- (N-morpholine) ethanesulfonic acid, preferably aqueous solution of 4-hydroxyethyl piperazine ethanesulfonic acid or 2- (N-morpholine) ethanesulfonic acid;
the myeloperoxidase detection reagent comprises oxidase, a substrate corresponding to the oxidase and a color developing agent A, wherein the oxidase and the substrate corresponding to the oxidase can generate hydrogen peroxide after meeting, and the color developing agent A comprises a Trinder reaction chromogen substrate;
the gingival protease detection reagent is a gingival protease detection reagent I or a gingival protease detection reagent II,
the gingival protease detection reagent I comprises a specific substrate BAEE, alcohol oxidase and a color-developing agent B, wherein the color-developing agent B comprises peroxidase and a Trinder reaction chromogen substrate, and the peroxidase is preferably horseradish peroxidase;
the gingival protease detection reagent II comprises a specific substrate BANA and a diazonium salt color developing agent.
2. The kit of claim 1, wherein: the Trinder reaction chromogen substrate is an oxidation coupling substrate pair or an oxidation reduction chromogen;
the oxidative coupling substrate pair is a substrate pair consisting of a substrate A and a substrate B, the substrate A is 4-aminoantipyrine or 3-methyl-2-benzothiazolinone hydrazone, the substrate B is selected from one of phenol, 3-hydroxy-2, 4, 6-tribromobenzoic acid, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline, N-ethyl-N- (3-sulfopropyl) -3-methylaniline, 3, 5-dichloro-2-sodium hydroxybenzenesulfonate, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline and N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline; preferably, the oxidative coupling substrate pair is a combination of 4-AAP and TBHBA, or a combination of MBTH and TBHBA;
the redox chromogen is selected from one of 3,3 '-diaminobenzidine, 3',5,5 '-tetramethylbenzidine, 3',5,5 '-tetramethylbenzidine dihydrochloride and 2,2' -dinitrogen-bis (3-ethylbenzothiazole-6-sulfonic acid), and is preferably TMB.2HCl.
3. The kit of claim 1, wherein: the oxidase in the myeloperoxidase detection reagent is one of glucose oxidase, alcohol oxidase, urate oxidase or cholesterol oxidase, and the substrate corresponding to the oxidase is glucose, alcohol, uric acid or cholesterol.
4. The kit of claim 1, wherein: the diazonium salt color developing agent is selected from fast blue B, fast blue BB salt, fast blue RR salt, fast purple B salt, fast red B, fast dark red GBC or fast black K salt, preferably fast black K salt.
5. The kit of claim 1, wherein: the concentration of the sample eluent is 20 mMol/L-50 mMol/L;
the dosage ratio of the oxidase to the substrate corresponding to the oxidase to the Trinder reaction chromogen substrate in the myeloperoxidase detection reagent is 0.1-0.5U/0.25-1 mg/0.1-1 mg;
the dosage ratio of specific substrates BAEE, ethanol oxidase, peroxidase and Trinder reaction chromogen substrate in the gingival protease detection reagent I is 0.25-1 mg/0.1-0.5U/0.02-0.2 mg/0.1-1 mg;
the dosage ratio of the specific substrate BANA to the diazonium salt color developing agent in the gingival protease detection reagent II is 0.25-1mg to 1.5-5 mg.
6. The kit of claim 1 or 5, wherein: the myeloperoxidase detection reagent and the gingiva protease detection reagent both further comprise trehalose and Proclin-300;
the dosage ratio of oxidase to trehalose in the myeloperoxidase detection reagent is 0.1-0.5U: 0.1-1 mg;
the dosage ratio of the specific substrate to the trehalose in the gingival protease detection reagent is 0.25-1 mg/0.1-1 mg.
7. The kit of any one of claims 2 to 6, wherein: the carrier of the kit comprises a microporous plate, a sample eluent bottle, a gingival protease chromogenic liquid bottle and a myeloperoxidase chromogenic liquid bottle,
the micro-pore plate is provided with at least one group of micro-pores, one group of micro-pores comprises two micro-pores, one micro-pore is coated with a gingival protease detection hole coating liquid I or a gingival protease detection hole coating liquid II, and the other micro-pore is coated with a myeloperoxidase detection hole coating liquid;
the coating liquid I for the gingival protease detection hole comprises: a specific substrate BAEE, alcohol oxidase, peroxidase, one of the substrates of an oxidation coupling substrate pair and deionized water; preferably, the composition further comprises trehalose;
the gingival protease chromogenic liquid bottle corresponding to the coating liquid I of the gingival protease detection hole contains: oxidizing the other substrate of the coupled substrate pair and deionized water; preferably also contains Proclin-300;
the coating liquid II for the gingival protease detection hole comprises: specific substrate BANA and deionized water; preferably, the composition further comprises trehalose;
the gingival protease chromogenic liquid bottle corresponding to the coating liquid II of the gingival protease detection hole contains: diazonium salt color developing agent and deionized water; preferably also contains Proclin-300;
the myeloperoxidase detection hole coating solution contains: oxidase, one of the substrates of the oxidative coupling substrate pair, and deionized water; preferably, the composition further comprises trehalose;
the myeloperoxidase chromogenic solution bottle contains: a substrate corresponding to oxidase, another substrate of an oxidative coupling substrate pair and deionized water; preferably also contains Proclin-300;
the sample eluent bottle contains a sample eluent.
8. The kit of claim 7, wherein: the micro-pore of the gum protease detection hole coating liquid I is coated on the micro-pore plate with the following coating: specific substrate BAEE 0.25-1 mg/hole, alcohol oxidase 0.1-0.5U/hole, peroxidase 0.02-0.2 mg/hole, one of the substrates of oxidative coupling substrate pair 0.1-1 mg/hole, trehalose 0-1 mg/hole; the gingival protease chromogenic solution bottle corresponding to the coating liquid I of the gingival protease detection hole contains the following components in concentration: 0.05-0.15g/L of the other substrate of the oxidative coupling substrate pair, 0-0.03ml/L of Proclin-300;
the micro-pores of the micro-pore plate coated with the gingival protease detection hole coating liquid II are coated with specific substrates BANA0.25-1 mg/hole and trehalose 0-1 mg/hole; the gingival protease chromogenic solution bottle corresponding to the gingival protease detection hole coating liquid II contains the following components in concentration: 30mg-100mg/L of diazonium salt color developing agent and 0-0.03ml/L of Proclin-300;
the micro-pores of the micro-pore plate coated with the myeloperoxidase detection hole coating liquid are coated with: 0.1-0.5U/hole of oxidase, 0.1-1 mg/hole of one of substrates of oxidative coupling substrate pair, and 0-1 mg/hole of trehalose; the myeloperoxidase chromogenic solution bottle contains the following components in concentration: 10-30g/L of substrate corresponding to oxidase, 0.05-0.15g/L of other substrate of oxidative coupling substrate pair, and 0-0.03ml/L of Proclin-300;
the concentration of the sample eluent is 20 mMol/L-50 mMol/L;
preferably, the concentration of the Proclin-300 is 0.01-0.03ml/L, and the concentration of the trehalose is 0.1-1 mg/hole.
9. The kit of claim 8, wherein: the volume of the micropores on the micropore plate is 100-150 ul.
10. A method for detecting a marker of periodontal pathogen infection, comprising: the kit of any one of claims 7 to 9 is used for detection, a sample to be detected of dental plaque or gingival crevicular fluid is collected, the sample to be detected is eluted from the surface of a sampling tool by using a sample eluent to obtain an eluted sample, the eluted sample is added into micropores on a micropore plate, then a developing solution in a developing solution bottle is added for reaction for 10 to 50 minutes to develop color, and whether the sample to be detected contains gingival protease and myeloperoxidase can be judged according to color change;
preferably, the amount of the sample eluent is 150-200 ul/per sample, and the amount of the color developing solution is 30 ul/well.
Technical Field
The invention relates to the technical field of biological and medical detection, in particular to a detection kit and a detection method for a periodontal pathogen infection marker.
Background
Alzheimer's Disease (AD) is a central neurodegenerative disease, accounting for 60-70% of senile dementia. The clinical manifestations are the neuropsychiatric symptoms such as progressive memory disorder, cognitive dysfunction, personality change and language disorder. Characteristic pathological changes are extracellular senile plaques formed by deposition of beta amyloid protein (A beta), neurofibrillary tangles (NFT) in nerve cells formed by hyperphosphorylation of tau protein, and neuronal loss with gliocyte hyperplasia, and the like.
The exact etiology and pathogenesis of AD has not yet been fully elucidated, but it is well established that the development of AD is the result of a combination of genetic and environmental factors, infection is the major environmental factor, and inflammatory responses in the central nervous system play a key role in the development and progression of AD.
Infectious pathogens that are clearly associated with AD include viruses, bacteria, spirochetes, etc., of which the most important and well studied are periodontal pathogens, mainly including Porphyromonas gingivalis (Pg), Treponema denticola (Td), Aggregatibacter actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), etc., which are gram-negative anaerobes and normally colonize dental plaque in the form of a plaque biofilm. When infection occurs due to the breakdown of the periodontal local immune defense barrier, periodontal pathogens proliferate in large numbers, and various toxic products such as gingipain are released, which stimulate local neutrophil infiltration, destroy gingiva and periodontal tissues, and cause gingivitis and periodontitis (collectively referred to as periodontal disease). Pathogenic bacteria and their toxic products, extracellular enzymes released by neutrophils, inflammatory mediators, etc. can cause characteristic pathological changes in the central nervous system through various mechanisms. Thus, periodontal pathogenic bacterial infection is a promoter of the development of AD and a factor promoting the progression of disease. The control of periodontal pathogenic bacteria infection has great guiding significance for the formulation of measures for preventing and controlling the disease development of AD.
AD is usually clinically diagnosed by neuropsychiatric symptom characteristics, characteristic physical markers. For example, the diagnosis of AD is supported by the detection of a β and Tau proteins, the risk of familial early-onset AD is determined by the detection of specific genes (e.g., amyloid precursor protein gene (APP), presenilin 1, 2 gene mutations (PS1, PS2)), and the detection of apolipoprotein APOE4 gene serves as a basis for the diagnosis of sporadic AD.
Infection markers of periodontal pathogenic bacteria include two types, pathogenic bacteria markers and inflammation markers.
The pathogenic bacteria markers comprise pathogenic bacteria individuals, genes and toxic products. Pathogenic bacteria mainly include periodontal pathogenic bacteria such as Pg, Td, Aa, Pi, Fn, etc., and are detected by conventional culture method, but the number of infectious bacteria cannot be reflected; the specific gene of the pathogenic bacteria can be detected by PCR, but the number of the infectious bacteria cannot be reflected; the toxic products of pathogenic bacteria comprise lipopolysaccharide, protease, lectin A and the like, wherein gingipain (gingipain) is shared by periodontal pathogenic bacteria, can be detected by an enzyme chemical method and an immunological method, can be quantified, and can directly reflect the quantity of infectious bacteria.
Inflammatory markers include both neutrophils and inflammatory mediators. The neutrophil is special in shape, contains specific enzymes (such as elastase, alkaline phosphatase, myeloperoxidase, beta-glucosidase and collagenase (matrix metalloproteinase-8, MMP-8)), can be quantitatively detected by a morphological observation method, an enzyme chemistry method, an immunological method and the like, and the quantity of the neutrophil in a sample can directly reflect the severity of local inflammation. Inflammatory mediators include cytokines (e.g., interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), Interferon-gamma (IFN-gamma)), prostaglandin E2(prostaglandinE2, PGE2), etc., which are immunologically detectable, quantifiable, and indirectly reflect the degree of local inflammation.
The current laboratory examination of AD is only limited to the examination of characteristic physical markers, infection markers related to AD are not detected, the detection result only has a supporting function for the diagnosis of the disease, and the guidance significance is not provided for the preparation of treatment measures such as the prevention of the disease, the control of the disease development and the like.
Disclosure of Invention
An object of the present invention is to solve the above-mentioned problems occurring in the prior art, and to provide a detection kit for a marker of periodontal disease infection, comprising a sample eluent, a myeloperoxidase detection reagent, and a gingipain detection reagent,
the sample eluent is aqueous solution of tris, 3-morpholinopropanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), piperazine-N, N' -di (2-ethanesulfonic acid) or 2- (N-morpholino) ethanesulfonic acid (MES), preferably aqueous solution of 4-hydroxyethylpiperazine ethanesulfonic acid or 2- (N-morpholino) ethanesulfonic acid;
the myeloperoxidase detection reagent comprises oxidase, a substrate corresponding to the oxidase and a color developing agent A, wherein the oxidase and the substrate corresponding to the oxidase can generate hydrogen peroxide after meeting, and the color developing agent A comprises a Trinder reaction chromogen substrate;
the gingival protease detection reagent is a gingival protease detection reagent I or a gingival protease detection reagent II,
the gingival protease detection reagent I comprises a specific substrate N-benzoyl-L-arginine ethyl ester (BAEE), an alcohol oxidase and a color developing agent B, wherein the color developing agent B comprises a peroxidase and a Trinder reaction chromogen substrate, and preferably, the peroxidase is horseradish peroxidase (HRP);
the gingival protease detection reagent II comprises a specific substrate N-benzoyl-L-arginine naphthamide (BANA) and a diazonium salt color developing agent.
The Trinder reaction chromogen substrate is an oxidation coupling substrate pair or an oxidation reduction chromogen;
the oxidative coupling substrate pair is a substrate pair consisting of a substrate A and a substrate B, wherein the substrate A is 4-aminoantipyrine (4-AAP) or 3-methyl-2-benzothiazolinone hydrazone (MBTH), and the substrate B is selected from phenol, 3-hydroxy-2, 4, 6-tribromobenzoic acid (TBHBA), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline sodium salt (TOPS), 3, 5-dichloro-2-hydroxybenzene sodium salt (DHBS), N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline sodium salt monohydrate (ADPS), One of N- (2-hydroxy-3-sulfopropyl) -3-5-dimethoxyaniline sodium salt (HDAOS); preferably, the oxidative coupling substrate pair is a combination of 4-AAP and TBHBA, or a combination of MBTH and TBHBA;
the redox chromogen is selected from one of 3,3 '-Diaminobenzidine (DAB), 3',5,5 '-Tetramethylbenzidine (TMB), 3',5,5 '-tetramethylbenzidine dihydrochloride (TMB.2HCL), and 2,2' -diaza-bis (3-ethylbenzothiazole-6-sulfonic Acid) (ABTS), preferably TMB.2HCl.
Preferably, the oxidase in the myeloperoxidase detection reagent is one of glucose oxidase, alcohol oxidase, urate oxidase or cholesterol oxidase, and the substrate corresponding to the oxidase is glucose, alcohol, uric acid or cholesterol.
Preferably, the diazonium salt colour developer is selected from fast blue B, fast blue BB salt, fast blue RR salt, fast violet B salt, fast red B, fast deep red GBC or fast black K salt, preferably fast black K salt.
Preferably, the concentration of the sample eluent is 20 mMol/L-50 mMol/L;
the dosage ratio of the oxidase to the substrate corresponding to the oxidase to the Trinder reaction chromogen substrate in the myeloperoxidase detection reagent is 0.1-0.5U/0.25-1 mg/0.1-1 mg;
the dosage ratio of specific substrates BAEE, ethanol oxidase, peroxidase and Trinder reaction chromogen substrate in the gingival protease detection reagent I is 0.25-1 mg/0.1-0.5U/0.02-0.2 mg/0.1-1 mg;
the dosage ratio of the specific substrate BANA to the diazonium salt color developing agent in the gingival protease detection reagent II is 0.25-1mg to 1.5-5 mg.
Preferably, the myeloperoxidase detecting reagent and the gingipain detecting reagent both further comprise trehalose and Proclin-300;
the dosage ratio of oxidase to trehalose in the myeloperoxidase detection reagent is 0.1-0.5U: 0.1-1 mg;
the dosage ratio of the specific substrate to the trehalose in the gingival protease detection reagent is 0.25-1 mg/0.1-1 mg.
Preferably, the carrier of the kit comprises a micro-porous plate, a sample eluent bottle, a gingival protease chromogenic solution bottle and a myeloperoxidase chromogenic solution bottle,
the micro-pore plate is provided with at least one group of micro-pores, one group of micro-pores comprises two micro-pores, one micro-pore is coated with a gingival protease detection hole coating liquid I or a gingival protease detection hole coating liquid II, and the other micro-pore is coated with a myeloperoxidase detection hole coating liquid;
the coating liquid I for the gingival protease detection hole comprises: a specific substrate BAEE, alcohol oxidase, peroxidase, one of the substrates of an oxidation coupling substrate pair and deionized water; preferably, the composition further comprises trehalose;
the gingival protease chromogenic liquid bottle corresponding to the coating liquid I of the gingival protease detection hole contains: oxidizing the other substrate of the coupled substrate pair and deionized water; preferably also contains Proclin-300;
the coating liquid II for the gingival protease detection hole comprises: specific substrate BANA and deionized water; preferably, the composition further comprises trehalose;
the gingival protease chromogenic liquid bottle corresponding to the coating liquid II of the gingival protease detection hole contains: diazonium salt color developing agent and deionized water; preferably also contains Proclin-300;
the myeloperoxidase detection hole coating solution contains: oxidase, one of the substrates of the oxidative coupling substrate pair, and deionized water; preferably, the composition further comprises trehalose;
the myeloperoxidase chromogenic solution bottle contains: a substrate corresponding to oxidase, another substrate of an oxidative coupling substrate pair and deionized water; preferably also contains Proclin-300;
the sample eluent bottle contains a sample eluent.
In the above technical solution, the micro-wells of the micro-well plate coated with the coating solution I of the gingival protease detection well are coated with: specific substrate BAEE 0.25-1 mg/hole, alcohol oxidase 0.1-0.5U/hole, peroxidase 0.02-0.2 mg/hole, one of the substrates of oxidative coupling substrate pair 0.1-1 mg/hole, trehalose 0-1 mg/hole; the gingival protease chromogenic solution bottle corresponding to the coating liquid I of the gingival protease detection hole contains the following components in concentration: 0.05-0.15g/L of the other substrate of the oxidative coupling substrate pair, 0-0.03ml/L of Proclin-300;
the micro-pores of the micro-pore plate coated with the gingival protease detection hole coating liquid II are coated with specific substrates BANA0.25-1 mg/hole and trehalose 0-1 mg/hole; the gingival protease chromogenic solution bottle corresponding to the gingival protease detection hole coating liquid II contains the following components in concentration: 30mg-100mg/L of diazonium salt color developing agent and 0-0.03ml/L of Proclin-300;
the micro-pores of the micro-pore plate coated with the myeloperoxidase detection hole coating liquid are coated with: 0.1-0.5U/hole of oxidase, 0.1-1 mg/hole of one of substrates of oxidative coupling substrate pair, and 0-1 mg/hole of trehalose; the myeloperoxidase chromogenic solution bottle contains the following components in concentration: 10-30g/L of substrate corresponding to oxidase, 0.05-0.15g/L of other substrate of oxidative coupling substrate pair, and 0-0.03ml/L of Proclin-300;
the concentration of the sample eluent is 20 mMol/L-50 mMol/L;
preferably, the concentration of the Proclin-300 is 0.01-0.03ml/L, and the concentration of the trehalose is 0.1-1 mg/hole.
Preferably, the volume of the micropores in the microplate is 100-.
The invention also aims to provide a method for detecting the periodontal pathogen infection marker, which adopts the kit for detection, collects a sample to be detected of dental plaque or gingival crevicular fluid, uses a sample eluent to elute the sample to be detected from the surface of a sampling tool to obtain an eluted sample, adds the eluted sample into a micropore on a micropore plate, then adds a developing solution in a developing solution bottle to react for 10-50 minutes for developing,
judging whether the sample to be detected contains the gingival protease and the myeloperoxidase or not according to the color change;
preferably, the amount of the sample eluent is 150-200 ul/per sample, and the amount of the color developing solution is 30 ul/well.
The kit is designed according to the principle of an enzymatic chemical color development method, can be provided in a form of a combination of a micro-porous plate, a sample treatment liquid and a color development liquid, is used for joint detection of gingival protease and neutrophil specific enzyme of a gingival crevicular fluid or dental plaque sample, and can judge the relative concentration of a to-be-detected object through color change.
The principle of the enzymatic chemical color development method is as follows: catalyzing the specific substrate to be tested to decompose to generate a new product; the new product is coupled and developed with an indicating system or a diazonium salt system, and the color depth is in direct proportion to the concentration of the substance to be detected.
In the gingival protease detection reagent I, gingival protease in a sample to be detected reacts with a specific substrate BAEE to generate ethanol, the ethanol generates hydrogen peroxide under the action of ethanol oxidase, and the hydrogen peroxide and peroxidase enable a Trinder reaction chromogen substrate to develop color.
The reaction product of the gingipain in the sample to be detected in the gingipain detection reagent II and the specific substrate BANA promotes the color development of the diazonium salt color developing agent.
The oxidase in the myeloperoxidase detection reagent and the corresponding substrate meet to generate hydrogen peroxide, and the myeloperoxidase in the sample to be detected makes the Trinder reaction chromogen substrate develop color under the action of the hydrogen peroxide.
Trehalose is used as a stabilizing agent in the reagent, and Proclin-300 is used as a preservative.
The color development result can be expressed as blue, green, red, purple, gray, black or other colors which can be identified by naked eyes.
The number of the micropores of the microporous plate is set according to the number of detectable persons of the single plate, and can be 4-22 pores, which are respectively used for simultaneous detection of 1-10 persons, and the volume of the micropores can be 100-.
The invention has the beneficial effects that: the kit can simultaneously detect infection markers of two types of periodontal pathogenic bacteria, namely a pathogenic bacteria marker and an inflammation marker, can simultaneously detect whether the bacteria are infected and whether the bacteria are inflamed, judges whether the periodontal disease exists, and can judge the relative content of the pathogenic bacteria marker and the inflammation marker, thereby judging the severity of the periodontal disease, and the kit is very suitable for clinical rapid diagnosis of the periodontal disease. Through the detection of the pathogenic bacteria marker, the risk assessment of AD can be carried out, so that the early intervention can be carried out, and the occurrence of AD can be prevented; and can also evaluate the AD disease progress correlation index, make corresponding measures and prevent the disease deterioration.
Drawings
FIG. 1 is a graph showing the results of detection of Porphyromonas gingivalis by the kit of the present invention.
FIG. 2 is a graph showing the result of detection of neutrophil myeloperoxidase quality control enzyme-horseradish peroxidase by the kit of the present invention.
FIG. 3 is a graph showing the results of detection of a plaque sample from a periodontal disease patient by the kit of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental procedures in the following examples are conventional unless otherwise specified.
The main reagents are as follows:
N-benzoyl-L-arginine ethyl ester BAEE (CAS:2645-08-1)
N-benzoyl-L-arginine naphthamide BANA (CAS: 913-04-2)
4-aminoantipyrine 4-AAP (CAS:83-07-8)
3-methyl-2-benzothiazolinone hydrazone (MBTH) (CAS:1128-67-2)
3-hydroxy-2, 4, 6-tribromobenzoic acid (TBHBA) (CAS:14348-40-4)
Solid black K salt (CAS:27766-47-8)
Proclin-300 liquid biological preservative
HRP: horseradish peroxidase
MES 2- (N-morphine) ethanesulfonic acid (CAS:4432-31-9)
The above reagents are conventional in the art and are commercially available.
The strains used in the examples were: porphyromonas gingivalis strain, BNCC 337441, is commercially available.
The medium used in the examples was colombian blood plates.
The remaining reagents in the examples, if not indicated, were conventional in the art and were also commercially available.
Example 1
The following reagents were prepared for use in the assay experiments in the various examples of the present invention.
First, preparation of sample eluent
Preparing sample eluent according to the following formula
MES 4.26g
Deionized Water 100ml
Adjusting pH to 6.5, standing at 2-8 deg.C, and refrigerating.
Preparation of microporous coating of microporous plate and corresponding color developing solution
Preparing a detection hole coating solution and a corresponding developing solution as shown in table 1:
TABLE 1
The blank microporous plate is also called a reaction card and can be directly purchased from the market. The microporous plate consists of a bottom plate and a plate cover, wherein a plurality of grooves (namely micropores) for placing filter paper sheets are arranged on the bottom plate, the filter paper sheets are firstly arranged on the bottom plate before use, and the filter paper sheets are used for attaching coating liquid.
Coating the micropores of the microporous plate with a coating solution: when the coating liquid is used for coating the micropores, 10ul of coating liquid is added into each hole, then the coating liquid is frozen and dried in vacuum, and the aluminum foil bag is sealed and packaged and is placed for refrigeration at 2-8 ℃ for standby. Different microwells can be coated with different coating solutions to detect gingival proteases or myeloperoxidase.
The prepared micro-porous plate can be divided into a single project coating type and a combined project coating type. All micropores of the single-item coated type microporous plate are coated with the same coating solution (coating solution for detecting gingival protease or myeloperoxidase) and are used for tests of quality control strains and quality control enzymes in the embodiment; the 1 st and 3 rd wells of the combined project coated type microplate are coated with the coating liquid for detecting the gingipain and the 2 nd and 4 th wells are coated with the coating liquid for detecting the myeloperoxidase, and the combined project coated type microplate is used for the test in the clinical sample detection embodiment.
Example 2 detection of gingipain from quality control Strain-Porphyromonas gingivalis Strain
1. And (3) strain amplification:
a) positive strains: after the porphyromonas gingivalis strain is recovered, transferring the porphyromonas gingivalis strain to a Columbia blood agar plate, placing the plate in an anaerobic bag, and culturing for 5-7 days at a constant temperature of 37 ℃ until black colonies appear on the surface of a culture medium;
b) negative strains: after the recovery of the streptococcus A strain, transferring the streptococcus A strain to a Columbia blood agar plate, and culturing the streptococcus A strain at the constant temperature of 37 ℃ for 24-48 hours until grass green hemolytic colonies appear in a culture medium;
2. preparing 0.5M bacteria liquid with turbidity unit: respectively scraping positive strains and negative strains, putting the positive strains and the negative strains into sterile physiological saline, and adjusting the concentration to 0.5 McLeod turbidity units;
3. preparing bacterial liquids with different concentrations: corresponds to 1.5 x 10 in 0.5M turbidity units8CFU/ml calculation, dilution preparation 1 x 10 separately with sample eluent7、1*106、1*105、1*104、1*103、1*102、1*1010CFU/ml bacterial liquid;
4. and (3) detection: performing test with single-item coated microporous plate coated with gingipain detection hole coating solution I, adding 1.5 × 10 to 1-9 th holes8、1*107、1*106、1*105、1*104、1*103、1*102、1*1010CFU/ml bacterial liquid of 30ul is added, and then the developing liquid of 30ul corresponding to the coating liquid I of the gingival protease detection hole is added;
the constant temperature incubator at 4.37 ℃ is placed for 20min, and the color change of the micropores is observed.
The results show that except 0CFU/ml, the other micropores change from colorless to purple red, the color depth is in direct proportion to the bacterial liquid concentration, and the bacterial liquid concentration is 1 x 103A significant color change is visible to the naked eye at CFU/ml. The results are shown in FIG. 1, the number on the right side of the microwell represents the bacterial liquid concentration, and 0CFU/ml is a negative control.
EXAMPLE 3 detection of Gum protease quality control enzyme-Trypsin
1. Preparing a concentrated quality control solution: preparing 256u/ml trypsin solution by using the sample eluent;
2. preparing quality control products with different concentrations: diluting the concentrated quality control solution with the sample eluent to prepare quality control products with trypsin concentrations of 256u/ml, 128u/ml, 64u/ml, 32u/ml, 16u/ml, 8u/ml, 4u/ml, 2u/ml, 1u/ml and 0.5u/ml respectively;
3. carrying out tests by using a single-item coated micro-porous plate coated with a coating liquid II of a gingival protease detection hole, wherein 30ul of quality control products with the concentrations of 64u/ml, 32u/ml, 16u/ml, 8u/ml, 4u/ml, 2u/ml, 1u/ml, 0.5u/ml and 0u/ml are respectively added into the 1 st to 9 th holes, and 30ul of color development liquid corresponding to the coating liquid II of the gingival protease detection hole is added;
the constant temperature incubator at 4.37 ℃ is placed for 20min, and the color change of the micropores is observed.
The results show that except for the case that the color of the 0u/ml hole is not changed, the rest micropores are changed from orange red to blue black, the color depth is in direct proportion to the concentration of the trypsin, and the obvious color change can be seen by naked eyes when the enzyme concentration is 2 u/ml. Since trypsin and gingipain can cause the same color reaction by using the detection kit of the present invention, commercially available trypsin can be used as the quality control enzyme of the gingipain detection reagent of the present invention in production to detect whether the gingipain detection reagent of the present invention is effective.
EXAMPLE 4 detection of neutrophil myeloperoxidase quality control enzyme, Horseradish peroxidase
1. Preparing a concentrated quality control solution: preparing 256u/ml horseradish peroxidase solution by using a sample eluent;
2. preparing quality control products with different concentrations: diluting the concentrated quality control solution with sample eluent to prepare quality control substances with horseradish peroxidase concentrations of 256u/ml, 128u/ml, 64u/ml, 32u/ml, 16u/ml, 8u/ml, 4u/ml, 2u/ml, 1u/ml, 0.5u/ml and 0u/ml respectively;
3. carrying out tests by using a single-item coated micro-porous plate coated with the coating solution I of the myeloperoxidase detection hole, wherein 30ul of quality control products of 64u/ml, 32u/ml, 16u/ml, 8u/ml, 4u/ml, 2u/ml, 1u/ml, 0.5u/ml and 0u/ml are respectively added into the 1 st to 9 th micro-pores, and 30ul of color development solution corresponding to the coating solution I of the myeloperoxidase detection hole is added;
the constant temperature incubator at 4.37 ℃ is placed for 20min, and the color change of the micropores is observed.
As shown in FIG. 2, all the wells showed a change in color from colorless to bluish-purple, with the color intensity being proportional to the concentration of horseradish peroxidase, and a visible color change was observed at 1 u/ml. Since horseradish peroxidase and myeloperoxidase can cause the same color reaction by using the detection kit of the invention, in the production, a commercial horseradish peroxidase can be used as the quality control enzyme of the myeloperoxidase detection reagent of the invention to detect whether the myeloperoxidase detection reagent is effective or not.
EXAMPLE 5 detection of plaque samples in periodontal disease patients
1. A specialist collects a subgingival dental plaque sample of a patient with confirmed periodontal disease by using a sterile toothpick and puts the subgingival dental plaque sample into a sample tube;
2. adding 200ul of sample eluent, repeatedly rolling and washing the toothpick with the sample in the sample eluent in a vibration washing mode to enable dental plaque to fall off, and blowing and dispersing the dental plaque by using a suction nozzle of a washing liquid gun to obtain sample liquid to be detected;
3. and (3) detection: and (3) carrying out a test by using a combined project coated microporous plate of which the 1 st and 3 rd holes are coated with the gingival protease detection hole coating liquid I and the 2 nd and 4 th holes are coated with the myeloperoxidase detection hole coating liquid II, using a sample eluent without a collected sample as a control, adding 30ul of sample liquid to be detected or the control into each micropore, and then adding 30ul of corresponding color development liquid. Placing in a constant temperature incubator at 37 deg.C for 20min, and observing the color change of the micropores.
As shown in FIG. 3, the periodontal disease patient sample liquid appeared reddish purple in the gingival protease detection wells (1 st and 3 rd wells), blue in the myeloperoxidase detection wells (2 nd and 4 th wells), and no color change occurred in the non-periodontal disease patient sample liquid (marked with S3 in FIG. 3) and the control well.
Example 6 clinical sample assay
36 parts of hospital clinical subgingival plaque samples are taken, wherein 26 parts of samples with periodontal disease and 10 parts of samples with other oral diseases are diagnosed. The microplate kit disclosed by the invention is used for detecting the 36 samples, the microplate is coated with a gingival protease detection hole coating solution I and a myeloperoxidase detection hole coating solution I, every two micropores form a group of detection holes, one micropore is coated with the gingival protease detection hole coating solution I, and the other micropore is coated with the myeloperoxidase detection hole coating solution I.
And (3) putting the subgingival dental plaque sample into a sample tube, adding 150ul of sample eluent, repeatedly rolling and washing the toothpick with the sample in the sample liquid to enable dental plaque to fall off, and blowing and dispersing the dental plaque by using a washing liquid gun suction nozzle to obtain the sample liquid to be detected. Adding 30ul of sample liquid to be detected into the micropores coated with the gingival protease detection hole coating liquid I, adding 30ul of sample liquid to be detected into the micropores coated with the myeloperoxidase detection hole coating liquid I, placing in a constant-temperature incubator at 37 ℃ for 20min, and observing the color change of the two micropores.
The results show that: the color of the gingival protease detection hole of the two micropores of each group of detection holes changes from colorless to purple/purple and the color of the myeloperoxidase detection hole changes from colorless to bluish purple, which shows that both gingival protease and myeloperoxidase in the sample are detected as positive. In addition, in 10 samples of other oral diseases, the color of the gingival protease detection micropore is not changed, 3 parts of myeloperoxidase detection pores are developed, and the neutrophilic granulocyte is found by microscopic examination of the developed sample.
The detection result of the kit conforms to the clinical result, which indicates that the kit can be applied to the simultaneous detection of the gingipain and the myeloperoxidase, thereby determining whether the periodontal disease is caused, and also judging the severity of the infection according to the depth of the color, and because the infection of the periodontal disease is a starting factor of AD occurrence and a promotion factor of disease deterioration, the kit can also be used for carrying out the risk assessment of AD occurrence by the detection of the kit, so as to intervene in advance and prevent the AD occurrence; and can also evaluate the AD disease progress correlation index, make corresponding measures and prevent the disease deterioration.
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