CN106399459A - Vaginal secretion detection kit and preparation method thereof - Google Patents
Vaginal secretion detection kit and preparation method thereof Download PDFInfo
- Publication number
- CN106399459A CN106399459A CN201610882226.8A CN201610882226A CN106399459A CN 106399459 A CN106399459 A CN 106399459A CN 201610882226 A CN201610882226 A CN 201610882226A CN 106399459 A CN106399459 A CN 106399459A
- Authority
- CN
- China
- Prior art keywords
- soak
- detection kit
- beta
- reacting
- coagulase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/10—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases p-Nitrophenol derivatives
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- C12Q2334/50—Indoles
- C12Q2334/52—5-Bromo-4-chloro-3-indolyl, i.e. BCI
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/10—Anilides
- C12Q2337/12—Para-Nitroanilides p-NA
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
- C12Q2337/30—Naphthyl amides, e.g. beta-NA, 2-NA, 4-methoxy-beta-naphthylamine, i.e. 4MNA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
- G01N2333/942—Hydrolases (3) acting on glycosyl compounds (3.2) acting on beta-1, 4-glucosidic bonds, e.g. cellulase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/952—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from bacteria
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Neurosurgery (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a vaginal fluid detection kit, which comprises a reaction device and a color developing agent, wherein the reaction device comprises a reaction hole for detecting beta-glucuronidase, a reaction hole for detecting acetylglucosaminidase, a reaction hole for detecting coagulase and a reaction hole for detecting proline aminopeptidase; when the kit is used for detecting vaginal secretion, the used diluent is normal saline. The vaginal secretion detection kit has the advantage that pathogenic bacteria or pathogens of bacterial vaginal diseases and trichomonas vaginitis can be used for assisting in the fast judgment of the vaginal disease condition. The invention also discloses a preparation method of the vaginal secretion detection kit.
Description
Technical field
The present invention relates to a kind of vaginal fluid detection kit and preparation method thereof, belong to vagina disease detection articles for use
Field.
Background technology
Vaginitis is the inflammation of connective tissue under vaginal mucosa and mucous membrane, is the common disease of Out-patient Clinic of Department of Gynecology.Normal health
Women, due to anatomy and biochemical characteristics, vagina has natural defense function to the intrusion of pathogen, when naturally preventing of vagina
Imperial function is destroyed, then pathogen is easy to invade, and leads to colpitis.Common vaginitis has bacterial vaginitis (BV/
AV), colpomycosis (VVC) and trichomonas vaginitis (TV) three major types.Wherein, women Bacterial leaf steak can be divided into two
Kind, one kind is caused by anaerobic bacteria and facultative anaerobic bacteria, clinically claims bacterial vaginosis BV (BV), and another kind is caused by aerobic bacteria,
Clinically claim aerobic bacteria property vaginitis (AV).The pathogen of two kinds of bacterial vaginitis is different, clinical symptoms, therapeutic scheme and knot
Office also differs, and distinguishes both Bacterial leaf steak, significant in the diagnosis and treatment of vagina infection.BV refers to a class
In genesiology, normal flora (produces H2O2Bacillus acidi lactici) reduce, instead Gardnerella, anaerobic bacteria, Mobiluncus amount reproduction,
The class disease that the change of vagina ecological balanced system causes.AV is that per vaginam Bacillus acidi lactici reduces, and aerobic bacteria infection causes
Colpitis.Common causative is streptococcus, staphylococcus and EHEC.Normal vagina is with Bacillus acidi lactici as advantage
The normal microflora of bacterium, vagina pH ＜ 4.5.And when suffering from aerobic bacteria property vaginitis, Vaginal lactobacilli reduces, and aerobic bacteria
Breeding leads to the hyperemia of vaginal wall mucous membrane, oedema and produces purulent secretion.Clinical manifestation with vagina abnormal secretion, intercourse pain is
Main.Trichomonas vaginitis (TV) is the class vaginal disease being produced for main pathogens with trichomonas vaginalis, is global, very
Common public health sexually transmitted disease.Bacterium preformation enzyme is a series of and growth generation that bacterium synthesizes during growth and breeding
Thank to relevant enzyme, these enzymes can decompose the substrate of multiple biochemical reactions, and can protect for a long time in aerobic environment
Stay its activity.The bacterium of different genera, the species of its preformation enzyme is different.B race streptococcus and EHEC metabolism can produce β Portugal
Grape uronic acid enzyme, trichomonad, candida albicans have 2-Acetamido-2-deoxy-D-glucose glycosides enzyme spcificity enzyme, the Main Pathogenic Bacteria yellow grape of AV
Coccus, enterococcus faecalis, EHEC can produce coagulase, and Prolyl iminopeptidase is to be produced by Gardnerella and Mobiluncus
Raw, it is the specificity marker enzyme secreted by BV correlation pathogen, so detecting to the carrying out of above enzyme, to diagnose flora in vagina
Structure is an effective, feasible method.But in prior art, there is not the kit that can simultaneously detect above four kinds of enzymes.
Content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the present invention is to provide a kind of vaginal secretion analyte detection examination
Agent box, this vaginal fluid detection kit can be with the pathogenic bacteria of detection bacterium vaginosis and trichomonas vaginitis or cause of disease
Body, auxiliary quickly judges vaginopathy situation.
Realize the purpose of the present invention to reach by adopting the following technical scheme that：A kind of vaginal fluid detection reagent
Box, including：Reaction unit, developer；Described reaction unit include for detect beta-glucuronidase enzyme reacting hole, be used for
Detect the reacting hole of acetyl glucosaminidase, be used for detecting the reacting hole of coagulase and being used for detecting Prolyl iminopeptidase
Reacting hole；During described kit detection during vaginal fluid, the dilution of employing is physiological saline.
Preferably, described developer includes the first developer with the reacting hole cooperation for detecting coagulase and uses
The second developer in the reacting hole cooperation of detection acetyl glucosaminidase；Described first developer is the hydrochloric acid of cinnamic acid
Solution；Described second developer is sodium hydroxide solution.
Preferably, the described reacting hole for detecting beta-glucuronidase enzyme is coated with the inspection of beta-glucuronidase enzyme
Test agent；Described beta-glucuronidase enzyme detection reagent includes：The bromo- 4- of polyvinylpyrrolidone, 5- chloro- 3- indoles-β-D-
Glucuronide salt, NBCS and phosphate buffer.
More preferably, the composition of described beta-glucuronidase enzyme detection reagent includes by weight：
1～10 part of polyvinylpyrrolidone；
The bromo- 4- of 5- chloro- 3- indoles-β -2.5～25 parts of D-Glucose aldehydic acid glycosides salt；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L.
Preferably, the described reacting hole for detecting acetyl glucosaminidase is coated with acetylglucosamine glycosides
Enzyme detection reagent；Described acetyl glucosaminidase detection reagent includes：Absolute methanol, p-nitrophenyl-N- acetylamino Portugal
Polyglycoside enzyme, polyvinylpyrrolidone, Triton X-100, EDTA and citric acid.
More preferably, the composition of described acetyl glucosaminidase detection reagent includes by weight：
The percent by volume that described Triton X-10 accounts for acetyl glucosaminidase detection reagent is 0.01～0.5%.
Preferably, the described reacting hole for detecting coagulase is coated with coagulase detection reagent；Described coagulase inspection
Test agent includes：Morpholino b acid buffer solution, sucrose, polyvinylpyrrolidone and glycyl-arginyl tetramethoxy-β-naphthalene
Amine.
More preferably, the composition of described coagulase detection reagent includes by weight：
0.2～10 part of sucrose；
0.2～2 part of polyvinylpyrrolidone；
Glycyl-arginyl tetramethoxy -0.2～2 part of beta-naphthylamine；
Wherein, the molar concentration of morpholino b acid buffer solution is：0.05～1.5mol/L.
Preferably, the described reacting hole for detecting Prolyl iminopeptidase is coated with Prolyl iminopeptidase detection examination
Agent；Described Prolyl iminopeptidase detection reagent includes：Dithiothreitol (DTT), sucrose, polyvinylpyrrolidone, Z- glycyl-dried meat
Propylhomoserin-paranitroanilinum, bovine serum albumin(BSA) and Tris-Hcl buffer solution.
More preferably, the composition of described Prolyl iminopeptidase detection reagent includes by weight：
Wherein, bovine serum albumin(BSA) accounts for the percent by volume 0.5～10% of Prolyl iminopeptidase detection reagent；
The molar concentration of Tris-Hcl buffer solution is 0.01～1mol/L.
Second object of the present invention is to provide for a kind of preparation method of detection kit, can be obtained by the method
Good stability, the high detection kit of accuracy.
Realize the purpose of the present invention to reach by adopting the following technical scheme that：A kind of detection kit as above
Preparation method, comprise the following steps：
1) prepare beta-glucuronidase enzyme reaction pad：By polyvinylpyrrolidone and the bromo- 4- of 5- chloro- 3- indoles-β-D-
Glucuronide salt is dissolved in phosphate buffer, obtains soak A；NBCS is substantially soluble in phosphate buffer
In, obtain soak B；Carrier is placed in soak A and fully soaks, then take out drying, then be placed in soak B and fully soak
Bubble, then takes out drying, obtains beta-glucuronidase enzyme reaction pad；
2) prepare acetyl glucosaminidase reacting pad：Will be fully molten for p-nitrophenyl -2-Acetamido-2-deoxy-D-glucose glycosides enzyme
In absolute methanol, obtain soak A；Polyvinylpyrrolidone, Triton X-10, EDTA and citric acid are dissolved in pure water,
Obtain soak B；Carrier is placed in soak A and fully soaks, then take out drying, then be placed in soak B and fully soak,
Then take out drying, obtain acetyl glucosaminidase reacting pad；
3) prepare coagulase reacting pad：By sucrose, polyvinylpyrrolidone and glycyl-arginyl tetramethoxy-β-naphthalene
Amine is dissolved in morpholino b acid buffer solution, obtains soak；Carrier is placed in soak and fully soaks, then take out drying,
Obtain coagulase reacting pad；
4) prepare Prolyl iminopeptidase reacting pad：By dithiothreitol (DTT), sucrose, polyvinylpyrrolidone, Z- glycyl-
Proline-para-nitroanilide and bovine serum albumin(BSA) are dissolved in Tris-Hcl buffer solution, obtain soak；Carrier is placed in immersion
Fully soak in liquid, then take out drying, obtain Prolyl iminopeptidase reacting pad；
5) prepare developer：Cinnamic acid is dissolved in hydrochloric acid solution, obtains the first developer；NaOH is dissolved in water
In, obtain the second developer；
6) placing response pad：By step 1)～4) the beta-glucuronidase enzyme reaction pad that obtains, acetylglucosamine
Glycosides enzyme reaction pad, coagulase reacting pad and Prolyl iminopeptidase reacting pad are respectively put in the corresponding hole position of kit, obtain
Reaction unit；
7) reaction unit is combined Ji Wei detection kit with developer.
Preferably, described step 1) in, the pH of phosphate buffer is 6.8～7.5.
Preferably, described step 1)～4) in, the time that carrier soaks each time in soak is 10～20s.
Preferably, described step 1)～4) in, the condition being dried each time is 20～75 DEG C of temperature, time 30～
40min.
The design principle of the present invention is as follows：
The reacting hole for detecting beta-glucuronidase enzyme in the present invention is coated with the detection of beta-glucuronidase enzyme
Reagent, this beta-glucuronidase enzyme detection reagent contains the bromo- 4- of 5- chloro- 3- indoles-β-D-Glucose aldehydic acid glycosides salt, works as vagina
When glucuronidase activity in secretion is abnormal, glucuronidase hydrolyzes the bromo- 4- of 5- chloro- 3- indoles-β-D- Portugal
Grape glycuronide salt, indyl assumes blueness, the activity of the depth of colour generation and beta-glucuronidase enzyme under conditions of aerobic
It is directly proportional；NBCS auxiliary strengthens the stability of beta-glucuronidase enzymatic activity so that testing result is accurate；
It is coated with acetylglucosamine glycosides in the reacting hole for detecting acetyl glucosaminidase in the present invention
Enzyme detection reagent, this acetyl glucosaminidase detection reagent contains p-nitrophenyl -2-Acetamido-2-deoxy-D-glucose glycosides enzyme, works as the moon
When there is in road secretion trichomonad, candida albicans, enzyme-specific can be produced：2-Acetamido-2-deoxy-D-glucose glycosides enzyme, hydrolysis p-nitrophenyl-
2-Acetamido-2-deoxy-D-glucose glycosides enzyme, displaing yellow in the presence of developer.Wherein, candida albicans leukorrhea pH≤4.6, and trichomonad leukorrhea
The detection kit of the present invention also can be differentiated trichomonas vaginitis or beads with reference to pH testing result by pH >=4.8 further
Bacterium property vaginitis；
The reacting hole for detecting coagulase in the present invention is coated with coagulase detection reagent；This coagulase detection reagent
Containing glycyl-arginyl tetramethoxy-beta-naphthylamine, when the solidification enzymatic activity in vaginal fluid is abnormal, solidify enzyme hydrolysis
Glycyl-arginyl tetramethoxy-beta-naphthylamine, aobvious light red, rose or reddish violet, colour generation in the presence of the first developer
Depth to solidification enzymatic activity be directly proportional；
Reacting hole for detecting Prolyl iminopeptidase is coated with Prolyl iminopeptidase detection reagent；This proline ammonia
Base peptase detection reagent contains Z- glycyl-proline-para-nitroanilide, when in vaginal fluid, normal flora (produces H2O2Lactic acid
Bacillus) reduce, instead Gardnerella, anaerobic bacteria, during Mobiluncus amount reproduction, Gardnerella and Mobiluncus produce dried meat ammonia
Sour aminopeptidase, Prolyl iminopeptidase hydrolyzes Z- glycyl-proline-para-nitroanilide, and paranitroanilinum assumes yellow；Two
Sulphur threitol and bovine serum albumin(BSA) auxiliary strengthen the stability of Prolyl iminopeptidase activity so that testing result is accurate.
Compared to existing technology, the beneficial effects of the present invention is：
1st, the detection kit of the present invention can zymosthenic stability, detection more accurate.
2nd, the detection kit of the present invention is used physiological saline as the dilution of detection sample, and use condition is simply square
Just, the sample after dilution is suitable for being detected with each reacting pad simultaneously and does not conflict, in hgher efficiency.
3rd, the detection reagent box preparation method of the present invention to greatest extent detection substrate composition is coated in reacting hole, obtains
The detection kit sensitivity obtaining is high, and accuracy is good.
4th, the detection kit of the present invention can detect beta-glucuronidase enzyme, acetyl glucosaminidase simultaneously, coagulate
Gu enzyme, Prolyl iminopeptidase.
Specific embodiment
Below, in conjunction with specific embodiment, the present invention is described further：
A kind of vaginal fluid detection kit, including：Reaction unit, developer；Described reaction unit is included for examining
Survey the reacting hole of beta-glucuronidase enzyme, be used for detecting the reacting hole of acetyl glucosaminidase, being used for detecting coagulase
Reacting hole and for detecting the reacting hole of Prolyl iminopeptidase；During described kit detection during vaginal fluid, employing
Dilution is physiological saline.
Described developer includes the first developer with the reacting hole cooperation for detecting coagulase and is used for detecting acetyl
Second developer of the reacting hole cooperation of UNAG；Described first developer is the hydrochloric acid solution of cinnamic acid；Described
Second developer is sodium hydroxide solution；
Wherein, the reacting hole for detecting beta-glucuronidase enzyme is coated with beta-glucuronidase enzyme detection reagent；
The composition of beta-glucuronidase enzyme detection reagent includes by weight：
1～10 part of polyvinylpyrrolidone；
The bromo- 4- of 5- chloro- 3- indoles-β -2.5～25 parts of D-Glucose aldehydic acid glycosides salt；
The percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L.
Reacting hole for detecting acetyl glucosaminidase is coated with acetyl glucosaminidase detection reagent；Institute
The composition stating acetyl glucosaminidase detection reagent includes by weight：
The percent by volume that described Triton X-10 accounts for acetyl glucosaminidase detection reagent is 0.01～0.5%.
The described reacting hole for detecting coagulase is coated with coagulase detection reagent；The one-tenth of described coagulase detection reagent
Divide and include by weight：
The described reacting hole for detecting Prolyl iminopeptidase is coated with Prolyl iminopeptidase detection reagent；Described dried meat
The composition of histidine amino group peptase detection reagent includes by weight：
Bovine serum albumin(BSA) accounts for the percent by volume 0.5～10% of Prolyl iminopeptidase detection reagent；
The molar concentration of Tris-Hcl buffer solution is 0.01～1mol/L.
Polyvinylpyrrolidone (PVP)-K90, at 25 DEG C, percentage by volume is 5% aqueous solution (aq), and its viscosity is
39.5～45.8mPas.
The preparation method of above-mentioned detection kit, comprises the following steps：
1) prepare beta-glucuronidase enzyme reaction pad：By polyvinylpyrrolidone and the bromo- 4- of 5- chloro- 3- indoles-β-D-
Glucuronide salt is dissolved in the phosphate buffer that pH is 6.8～7.5, obtains soak A；NBCS is fully molten
In pH be 6.8～7.5 phosphate buffer in, obtain soak B；By carrier be placed in soak A abundant soak 10～
20s, then takes out and is 40 DEG C in temperature, and the time is to be dried under conditions of 30～40min, then is placed in soak B and fully soaks
10～20s, then takes out and is 40 DEG C in temperature, and the time, for being dried under conditions of 30～40min, obtains beta-glucuronidase enzyme
Reacting pad；
Described phosphate buffer (PBS) is NaH2PO4And Na2HPO4The aqueous solution；
2) prepare acetyl glucosaminidase reacting pad：Will be fully molten for p-nitrophenyl -2-Acetamido-2-deoxy-D-glucose glycosides enzyme
In absolute methanol, obtain soak A；Polyvinylpyrrolidone, Triton X-10, EDTA and citric acid are dissolved in pure water,
Obtain soak B；Carrier is placed in soak A abundant immersion 10～20s, then takes out and be 40 DEG C in temperature, the time is 30
It is dried under conditions of～40min, then is placed in soak B abundant immersion 10～20s, then take out and be 45 DEG C in temperature, the time
It is dried under conditions of 30～40min, obtain acetyl glucosaminidase reacting pad；
3) prepare coagulase reacting pad：By sucrose, polyvinylpyrrolidone and glycyl-arginyl tetramethoxy-β-naphthalene
Amine is dissolved in morpholino b acid buffer solution (MES), obtains soak；Carrier is placed in soak abundant immersion 10～20s, so
After take out temperature be 40 DEG C, the time be 30～40min under conditions of be dried, obtain coagulase reacting pad；
4) prepare Prolyl iminopeptidase reacting pad：By dithiothreitol (DTT), sucrose, polyvinylpyrrolidone, Z- glycyl-
Proline-para-nitroanilide and bovine serum albumin(BSA) are dissolved in Tris-Hcl buffer solution, obtain soak；Carrier is placed in immersion
Fully soak 10～20s in liquid, then take out and be 45 DEG C in temperature, be dried under conditions of time 30～40min, obtain proline
Aminopeptidase reacting pad；
5) prepare developer：Cinnamic acid is dissolved in hydrochloric acid solution, obtains the first developer；NaOH is dissolved in water
In, obtain the second developer；The hydrochloric acid solution molar concentration of the first developer is 0.5～2mol/L, and cinnamic acid is in hydrochloric acid solution
In concentration be 5～13g/L；The naoh concentration of the second developer is 0.004～0.04g/mL；
6) by step 1)～4) the beta-glucuronidase enzyme reaction pad that obtains, acetyl glucosaminidase reacting pad,
Coagulase reacting pad and Prolyl iminopeptidase reacting pad are respectively put in the corresponding hole position of kit, obtain reaction unit；
7) reaction unit is combined Ji Wei detection kit with developer；Detection kit is placed in 2～8 DEG C of preservations.
Wherein carrier can be filter paper, more specifically, described filter paper can be Whatman glass fiber filter paper, GF/A
The fiber filter paper Grade3 or common quantitative filter paper of Whatman.
Embodiment 1～3
Embodiment 1～3 disclose for detect beta-glucuronidase enzyme reacting hole, be used for detecting acetamido glucose
The reacting hole of glycosidase, for detecting the reacting hole of coagulase and for detecting the detection in the reacting hole of Prolyl iminopeptidase
Agent formulations, specifically as shown in table 1, prepare solution according to the parameter shown in table 2, according to parameter preparation first colour developing in table 3
Agent and the second developer；And detection kit is prepared by the method in specific embodiment：
The material composition of table 1 embodiment 1～3 and content
The preparation parameter of table 2 solution
Table 3 first developer and the preparation parameter of the second developer
The detection kit obtaining in embodiment 1～3 is detected respectively：Prepare respectively the different β of concentration of enzymatic activity-
Glucuronidase standard solution, acetyl glucosaminidase standard solution, coagulase standard solution and dried meat ammonia
Sour aminopeptidase standard solution；Standard solution all using physiological saline as solvent, detects the sensitivity of embodiment 1～3；
Beta-glucuronidase enzyme standard solution is dripped in the reacting hole for detecting beta-glucuronidase enzyme
35uL；
In the reacting hole for detecting acetyl glucosaminidase, dropping acetyl glucosaminidase standard items are molten
Liquid 35uL；
Coagulase standard solution 35uL is dripped in the reacting hole for detecting coagulase；
Prolyl iminopeptidase standard solution 35uL is dripped for detecting in the reacting hole of Prolyl iminopeptidase；
Detection kit is placed in water-bath 8～10min under conditions of temperature is 37 ± 2 DEG C, take out observation be used for detecting β-
The color of the reacting hole of glucuronidase；Face is observed after dripping the first developer in the reacting hole for detecting coagulase
Color；Again detection kit is placed under conditions of temperature is 37 ± 2 DEG C and continues water-bath 8～10min, then take out observation and be used for examining
Survey the color of the reacting hole of Prolyl iminopeptidase, the reacting hole for detecting acetyl glucosaminidase drips second
Color is observed, colour developing represents that testing result is as shown in table 5 as shown in table 4 after developer：
The colour developing of table 4 detection kit represents
| Detection project | Positive | Negative |
| Beta-glucuronidase enzyme | Light blue, blue or bluish violet | Do not develop the color |
| Acetyl glucosaminidase | Yellow | Do not develop the color |
| Coagulase | Light red, rose or reddish violet | Do not develop the color |
| Prolyl iminopeptidase | Yellow | Do not develop the color |
The testing result of table 5 embodiment 1～3
+ represent positive ,-represent negative.
Result shows, embodiment 1～3 is equal to detect beta-glucuronidase enzyme, acetyl glucosaminidase, coagulase
And Prolyl iminopeptidase, the wherein detection kit sensitivity highest of embodiment 1,3.75U/mL remain to detection obtain above
The presence of four kinds of enzymes.
Comparative example 1～2
Gather sample from human body, carried out with the detection kit of comparative example 1～2 using the detection kit of embodiment 1
Detection, comparative example takes from vaginal fluid detection kit on the market, and the testing result of embodiment 1 is as shown in table 6：
The testing result of table 6 embodiment 1
The testing result of comparative example 1 is as shown in table 7：
The testing result of table 7 comparative example 1
The testing result of comparative example 2 is as shown in table 8：
The testing result of table 8 comparative example 2
In conjunction with spss statistical analysis software, analyze kappa value, evaluate the uniformity of embodiment 1 and comparative example, statistical analysis
Result is as shown in table 9～14；The positive coincidence rate (kappa) of embodiment 1 and comparative example 1～2, as shown in Table 15：
Table 9 acetyl glucosaminidase enzyme detection data cross tabulation
Table 10 acetyl glucosaminidase enzyme detection data spss is analyzed
Table 11 coagulase detection data cross tabulation
Table 12 coagulase detection data spss is analyzed
Table 13 Prolyl iminopeptidase detection data cross tabulation
Table 14 Prolyl iminopeptidase detection data spss is analyzed
Table 15 embodiment 1 and the uniformity evaluation result of comparative example
0<Kappa value<0.4 indicates uniformity, but the degree of consistency is poor；
0.4≤kappa value<0.75 represents that the degree of consistency is general；
Kappa value >=0.75 represents that the degree of consistency is good；
Result shows, compares with similar detection kit, and similar Testing index sample results coincidence rate and positive rate are basic
Unanimously.
For a person skilled in the art, can technical scheme as described above and design, make other each
Plant corresponding change and deform, and all these changes and deforms the protection model that all should belong to the claims in the present invention
Within enclosing.
Claims (14)
1. a kind of vaginal fluid detection kit is it is characterised in that include：Reaction unit, developer；Described reaction unit bag
Include for detect beta-glucuronidase enzyme reacting hole, for detecting the reacting hole of acetyl glucosaminidase, be used for examining
Survey the reacting hole of coagulase and the reacting hole for detecting Prolyl iminopeptidase；Vaginal fluid during described kit detection
When, the dilution of employing is physiological saline.
2. vaginal fluid detection kit as claimed in claim 1 it is characterised in that：Described developer include with for examining
Survey first developer of reacting hole cooperation of coagulase and for detecting the reacting hole cooperation of acetyl glucosaminidase
Two developers；Described first developer is the hydrochloric acid solution of cinnamic acid；Described second developer is sodium hydroxide solution.
3. vaginal fluid detection kit as claimed in claim 1 it is characterised in that：Described for detecting β-grape alditol
The reacting hole of neuraminidase is coated with beta-glucuronidase enzyme detection reagent；Described beta-glucuronidase enzyme detection reagent bag
Include：The bromo- 4- of polyvinylpyrrolidone, 5- chloro- 3- indoles-β-D-Glucose aldehydic acid glycosides salt, NBCS and phosphate-buffered
Liquid.
4. vaginal fluid detection kit as claimed in claim 3 it is characterised in that：Described beta-glucuronidase enzyme inspection
The composition of test agent includes by weight：
1～10 part of polyvinylpyrrolidone；
The bromo- 4- of 5- chloro- 3- indoles-β -2.5～25 parts of D-Glucose aldehydic acid glycosides salt；
Wherein, the percent by volume that NBCS accounts for beta-glucuronidase enzyme detection reagent is 1～10%；
The molar concentration of phosphate buffer is 50～150mmol/L.
5. vaginal fluid detection kit as claimed in claim 1 it is characterised in that：Described for detecting acetylamino Portugal
The reacting hole of polyglycoside enzyme is coated with acetyl glucosaminidase detection reagent；Described acetyl glucosaminidase detection examination
Agent includes：Absolute methanol, p-nitrophenyl -2-Acetamido-2-deoxy-D-glucose glycosides enzyme, polyvinylpyrrolidone, Triton X-100,
EDTA and citric acid.
6. vaginal fluid detection kit as claimed in claim 5 it is characterised in that：Described acetyl glucosaminidase
The composition of detection reagent includes by weight：
The percent by volume that described Triton X-10 accounts for acetyl glucosaminidase detection reagent is 0.01～0.5%.
7. vaginal fluid detection kit as claimed in claim 1 it is characterised in that：Described for detecting the anti-of coagulase
Hole is answered to be coated with coagulase detection reagent；Described coagulase detection reagent includes：Morpholino b acid buffer solution, sucrose, polyethylene
Pyrrolidones and glycyl-arginyl tetramethoxy-beta-naphthylamine.
8. vaginal fluid detection kit as claimed in claim 7 it is characterised in that：The one-tenth of described coagulase detection reagent
Divide and include by weight：
0.2～10 part of sucrose；
0.2～2 part of polyvinylpyrrolidone；
Glycyl-arginyl tetramethoxy -0.2～2 part of beta-naphthylamine；
Wherein, the molar concentration of morpholino b acid buffer solution is：0.05～1.5mol/L.
9. vaginal fluid detection kit as claimed in claim 1 it is characterised in that：Described for detecting amino proline
The reacting hole of peptase is coated with Prolyl iminopeptidase detection reagent；Described Prolyl iminopeptidase detection reagent includes：Two sulphur
Threitol, sucrose, polyvinylpyrrolidone, Z- glycyl-proline-para-nitroanilide, bovine serum albumin(BSA) and Tris-Hcl
Buffer solution.
10. vaginal fluid detection kit as claimed in claim 9 it is characterised in that：Described Prolyl iminopeptidase inspection
The composition of test agent includes by weight：
Wherein, bovine serum albumin(BSA) accounts for the percent by volume 0.5～10% of Prolyl iminopeptidase detection reagent；
The molar concentration of Tris-Hcl buffer solution is 0.01～1mol/L.
A kind of 11. preparation methods of detection kit as claimed in claim 1 are it is characterised in that comprise the following steps：
1) prepare beta-glucuronidase enzyme reaction pad：By polyvinylpyrrolidone and the bromo- 4- of 5- chloro- 3- indoles-β-D- grape
Glycuronide salt is dissolved in phosphate buffer, obtains soak A；NBCS is substantially soluble in phosphate buffer,
Obtain soak B；Carrier is placed in soak A fully soak, then takes out drying, then be placed in soak B and fully soak, so
Take out drying afterwards, obtain beta-glucuronidase enzyme reaction pad；
2) prepare acetyl glucosaminidase reacting pad：P-nitrophenyl -2-Acetamido-2-deoxy-D-glucose glycosides enzyme is substantially soluble in no
In water methanol, obtain soak A；Polyvinylpyrrolidone, Triton X-10, EDTA and citric acid are dissolved in pure water, obtain
Soak B；Carrier is placed in soak A and fully soaks, then take out drying, then be placed in soak B and fully soak, then
Take out drying, obtain acetyl glucosaminidase reacting pad；
3) prepare coagulase reacting pad：Will be molten to sucrose, polyvinylpyrrolidone and glycyl-arginyl tetramethoxy-beta-naphthylamine
In morpholino b acid buffer solution, obtain soak；Carrier is placed in soak and fully soaks, then take out drying, obtain
Coagulase reacting pad；
4) prepare Prolyl iminopeptidase reacting pad：By dithiothreitol (DTT), sucrose, polyvinylpyrrolidone, Z- glycyl-dried meat ammonia
Acid-paranitroanilinum and ox blood is pure is dissolved in Tris-Hcl buffer solution, obtains soak；Carrier is placed in soak fully
Soak, then take out drying, obtain Prolyl iminopeptidase reacting pad；
5) prepare developer：Cinnamic acid is dissolved in hydrochloric acid solution, obtains the first developer；NaOH is soluble in water, obtain
To the second developer；
6) placing response pad：By step 1)～4) the beta-glucuronidase enzyme reaction pad that obtains, acetyl glucosaminidase
Reacting pad, coagulase reacting pad and Prolyl iminopeptidase reacting pad are respectively put in the corresponding hole position of kit, are reacted
Device；
7) reaction unit is combined Ji Wei detection kit with developer.
The preparation method of 12. detection kit as claimed in claim 11 it is characterised in that：Described step 1) in, phosphate
The pH of buffer solution is 6.8～7.5.
The preparation method of 13. detection kit as claimed in claim 11 it is characterised in that：Described step 1)～4) in, carry
The time that body soaks each time in soak is 10～20s.
The preparation method of 14. detection kit as claimed in claim 11 it is characterised in that：Described step 1)～4) in, often
The condition of primary drying is 20～75 DEG C of temperature, time 30～40min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610882226.8A CN106399459A (en) | 2016-09-30 | 2016-09-30 | Vaginal secretion detection kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610882226.8A CN106399459A (en) | 2016-09-30 | 2016-09-30 | Vaginal secretion detection kit and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106399459A true CN106399459A (en) | 2017-02-15 |
Family
ID=59228401
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610882226.8A Pending CN106399459A (en) | 2016-09-30 | 2016-09-30 | Vaginal secretion detection kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106399459A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107907682A (en) * | 2017-11-02 | 2018-04-13 | 北京中生金域诊断技术股份有限公司 | For distinguishing the detection kit and preparation method of gram- bacteria in vaginal fluid |
| CN109991218A (en) * | 2019-03-29 | 2019-07-09 | 迪瑞医疗科技股份有限公司 | A kind of preparation method of coagulase Test paper |
| CN110243817A (en) * | 2019-07-16 | 2019-09-17 | 迪瑞医疗科技股份有限公司 | A kind of GRD beta-glucuronidase drying chemical reagent paper and detection method |
| CN114317678A (en) * | 2021-12-31 | 2022-04-12 | 港龙生物技术（深圳）有限公司 | Biological paper chip, high-flux multi-connection detection microporous plate device, preparation method and kit for multi-connection detection of vaginitis |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792792A (en) * | 2009-12-30 | 2010-08-04 | 北京中生金域诊断技术有限公司 | Kit for detecting aerobic bacteria in vaginal discharge and preparation method thereof |
| CN102866248A (en) * | 2012-09-28 | 2013-01-09 | 北京鸿天志远科技有限公司 | Stabilizer solution and preparation method thereof, enzyme conjugate mixture and protein mixture |
| CN102864208A (en) * | 2012-08-31 | 2013-01-09 | 北京中生金域诊断技术有限公司 | Quality control product of AV/BV combined test kit and preparation method thereof |
| CN103472235A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting protein solution stabilizing agent |
| CN204666520U (en) * | 2015-05-29 | 2015-09-23 | 珠海丽珠试剂股份有限公司 | A kind of vaginopathy combined detection reaction plate |
| CN104946616A (en) * | 2015-05-12 | 2015-09-30 | 骏实生物科技(上海)有限公司 | General solid stabilizer used for in vitro diagnostic reagent and application method of general solid stabilizer |
| CN205333640U (en) * | 2016-01-29 | 2016-06-22 | 河南美凯生物科技有限公司 | Combined detection card for vaginitis |
-
2016
- 2016-09-30 CN CN201610882226.8A patent/CN106399459A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792792A (en) * | 2009-12-30 | 2010-08-04 | 北京中生金域诊断技术有限公司 | Kit for detecting aerobic bacteria in vaginal discharge and preparation method thereof |
| CN102864208A (en) * | 2012-08-31 | 2013-01-09 | 北京中生金域诊断技术有限公司 | Quality control product of AV/BV combined test kit and preparation method thereof |
| CN102866248A (en) * | 2012-09-28 | 2013-01-09 | 北京鸿天志远科技有限公司 | Stabilizer solution and preparation method thereof, enzyme conjugate mixture and protein mixture |
| CN103472235A (en) * | 2013-08-26 | 2013-12-25 | 河北省科学院生物研究所 | Long-acting protein solution stabilizing agent |
| CN104946616A (en) * | 2015-05-12 | 2015-09-30 | 骏实生物科技(上海)有限公司 | General solid stabilizer used for in vitro diagnostic reagent and application method of general solid stabilizer |
| CN204666520U (en) * | 2015-05-29 | 2015-09-23 | 珠海丽珠试剂股份有限公司 | A kind of vaginopathy combined detection reaction plate |
| CN205333640U (en) * | 2016-01-29 | 2016-06-22 | 河南美凯生物科技有限公司 | Combined detection card for vaginitis |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107907682A (en) * | 2017-11-02 | 2018-04-13 | 北京中生金域诊断技术股份有限公司 | For distinguishing the detection kit and preparation method of gram- bacteria in vaginal fluid |
| CN109991218A (en) * | 2019-03-29 | 2019-07-09 | 迪瑞医疗科技股份有限公司 | A kind of preparation method of coagulase Test paper |
| CN110243817A (en) * | 2019-07-16 | 2019-09-17 | 迪瑞医疗科技股份有限公司 | A kind of GRD beta-glucuronidase drying chemical reagent paper and detection method |
| CN114317678A (en) * | 2021-12-31 | 2022-04-12 | 港龙生物技术（深圳）有限公司 | Biological paper chip, high-flux multi-connection detection microporous plate device, preparation method and kit for multi-connection detection of vaginitis |
| CN114317678B (en) * | 2021-12-31 | 2023-11-10 | 港龙生物技术（深圳）有限公司 | Biological paper chip, high-throughput multi-detection microplate device, preparation method and kit for vaginal inflammation multi-detection |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106399459A (en) | Vaginal secretion detection kit and preparation method thereof | |
| CN207894938U (en) | Diazol method GRD beta-glucuronidase drying chemical reagent paper | |
| Hamann et al. | Teat thickness changes may provide biological test for effective pulsation | |
| CN102253206A (en) | Combined detection kit for vaginitis | |
| CN104792986B (en) | A kind of vaginitiss many index combined detection kit | |
| CN106442453A (en) | Method for rapidly and quantitatively measuring spore concentration | |
| CN101718793B (en) | Preparation method of Rh blood type typing card | |
| JP2008513001A (en) | Method for detecting Streptococcus agalactiae using α-glucosidase activity | |
| CN103266163B (en) | Combined detection kit for detecting and identifying candida and trichomonias | |
| CN106442989B (en) | A kind of Prolyl iminopeptidase detection reagent, reacting pad, preparation method and kit | |
| CN101792792A (en) | Kit for detecting aerobic bacteria in vaginal discharge and preparation method thereof | |
| CN106501248B (en) | A kind of method of urea in high-throughput enzyme sensor and detection human urine | |
| CN107607719A (en) | Beta-lactam enzyme method in a kind of determination sample | |
| CN106350571B (en) | A kind of method of the sensitive antibiotics of quick screening treatment mastitis for milk cows | |
| CN106645720A (en) | Beta-glucuronidase detection reagent, reaction pad, preparation method of reaction pad and kit | |
| CN109459428A (en) | A kind of lactate detection drying chemical reagent paper and preparation method thereof | |
| US5702911A (en) | Diagnostic test composition | |
| CN106645721B (en) | A kind of coagulase detection reagent, reacting pad, preparation method and kit | |
| CN105713955B (en) | A kind of B races streptococcus differential medium | |
| CN106319028A (en) | Kit for detecting mycoplasma urealytium and human mycoplasma | |
| CN111235213A (en) | Method for checking total aerobic bacteria number of ranitidine hydrochloride capsule | |
| CN102251021A (en) | Detection kit for bacterial vaginosis (BV) and sialidase detection method | |
| CN110243817A (en) | A kind of GRD beta-glucuronidase drying chemical reagent paper and detection method | |
| CN109991218A (en) | A kind of preparation method of coagulase Test paper | |
| CN113567217A (en) | Gynecological fluorescent staining solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination |