阅读说明：本技术 一种需氧菌性阴道炎检测试纸及其制备方法 (Aerobic vaginitis test paper and preparation method thereof ) 是由 蒋庆姣 刘云鹏 潘军全 粟传军 于 2021-03-17 设计创作，主要内容包括：本发明公开了一种需氧菌性阴道炎检测试纸及其制备方法,包括在反应基板上,设置多个载体,多个载体在保留一个空白块后,剩余载体通过分别浸润β-葡萄糖醛酸苷酶溶液、凝固酶溶液、过氧化氢溶液、白细胞酯酶溶液、pH值溶液后经干燥制得。其中PH值、白细胞酯酶、过氧化氢、β-葡萄糖醛酸苷酶及凝固酶五个项目,可全面检测评估需氧菌性阴道炎；一步法操作,检测无需加显色液或终止液,操作简单,避免误操作,有利于自动化检测；反应只需要孵育5分钟即可进行检测,检测时间短,缩短TAT时间,适合临床试验检测特别是医院门诊检测筛查。(The invention discloses an aerobic vaginitis test paper and a preparation method thereof, and the test paper comprises a plurality of carriers arranged on a reaction substrate, wherein after a blank block is reserved for the carriers, the rest carriers are respectively soaked in a beta-glucuronidase solution, a coagulase solution, a hydrogen peroxide solution, a leukocyte esterase solution and a pH value solution and then dried to obtain the aerobic vaginitis test paper. Wherein, the pH value, the leukocyte esterase, the hydrogen peroxide, the beta-glucuronidase and the coagulase can be comprehensively detected and evaluated for aerobic bacterial vaginitis; the one-step method is operated, the color developing solution or the stop solution is not required to be added for detection, the operation is simple, the misoperation is avoided, and the automatic detection is facilitated; the reaction can be detected only by incubating for 5 minutes, the detection time is short, the TAT time is shortened, and the kit is suitable for clinical test detection, particularly hospital outpatient detection screening.)

1. An aerobic vaginitis test paper, which is characterized in that,

the carrier-free preparation method comprises a reaction substrate and a plurality of carriers, wherein after a blank block is reserved for the plurality of carriers, the rest carriers are prepared by respectively soaking a beta-glucuronidase solution, a coagulase solution, a hydrogen peroxide solution, a leukocyte esterase solution and a pH value solution and then drying, and the plurality of carriers are fixedly arranged on the reaction substrate.

2. An aerobic vaginitis test strip according to claim 1, wherein,

the carrier is a test paper block or a reaction hole.

3. A method for preparing an aerobic vaginitis test strip, which is used for preparing the aerobic vaginitis test strip according to any one of claims 1 to 2,

the method comprises the following steps: preparing a beta-glucuronidase carrier;

preparing a coagulase carrier;

preparing a leukocyte esterase carrier;

preparing a hydrogen peroxide carrier;

preparing a pH value carrier;

all carriers were attached to the reaction substrate, respectively, to complete the preparation.

4. A method of making an aerobic vaginitis test strip according to claim 3 wherein,

the specific steps for preparing the beta-glucuronidase carrier are as follows:

dissolving a substrate in a buffer solution with the pH value of 4-9 and the concentration of 0.5-10 mM, adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction accelerator to form a solution A, wherein the substrate is one or more of 5-bromo-4-chloro-3-indole-beta-D-glucuronide sodium salt, 4-nitrophenyl-beta-D-glucopyranoside (PNPG), 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and phenolphthalein glucuronide, the buffer solution is PBS buffer solution, citric acid-trisodium citrate buffer solution, citric acid-TRIS buffer solution and boric acid-borax buffer solution, one of sodium acetate buffer solution, wherein the reaction promoter is one or more of magnesium chloride, zinc chloride, calcium chloride, glycol, sodium alginate, glutaraldehyde and sucrose;

dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5-10 g/L to form a solution B, wherein the diazonium salt color developing agent is one of 2-methoxy-4-morpholinyl benzene diazonium salt, 2-ethoxy-4-morpholinyl benzene diazonium salt and 4-methoxybenzyl diazonium salt;

mixing the solution A and the solution B according to a ratio of 1:1 to form a beta-glucuronidase solution;

the beta-glucuronidase solution is attached to the carrier and dried.

5. A method of making an aerobic vaginitis test strip according to claim 3 wherein,

the preparation method of the coagulase carrier comprises the following specific steps:

dissolving a substrate in a buffer solution with the pH value of 3-7 and the concentration of 0.1mM-5mM for buffering, and adding a stabilizer to form a solution A after dissolution is finished, wherein the substrate is glycyl-arginyl-4-methoxy-beta-naphthylamine, the stabilizer is a mixed solution of trehalose and sucrose, and the buffer solution is hydrochloric acid-TRIS buffer solution;

dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5-10 g/L to form a B solution, wherein the diazonium salt color developing agent is one of fast violet B salt, fast blue BB salt, fast blue RR salt and fast red B salt;

and soaking and drying the carrier in a coagulase A solution, then soaking in a coagulase B solution, and then drying to obtain the carrier.

6. A method of making an aerobic vaginitis test strip according to claim 3 wherein,

the preparation method of the leucocyte esterase carrier comprises the following specific steps:

dissolving a substrate in a buffer solution with the pH value of 6-9 and the concentration of 0.1-2 mM, adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction promoter to form solution A, wherein the substrate is indoxyl ester and derivatives thereof, one or more of pyrrole ester and pyrrole amino acid ester derivatives thereof, and the stabilizer is one or more of beta-cyclodextrin, mannitol, ethylene glycol, glycerol, sodium ethylene diamine tetraacetate and potassium ethylene diamine tetraacetate;

dissolving diazo salt developer in organic solvent containing 0.5-10 g/L organic acid and adding 0.1-5 g/L surfactant to form solution B;

mixing the solution A and the solution B according to the proportion of 1: 1-9: 1 to form a leukocyte esterase solution;

the carrier is soaked in leukocyte esterase solution and then dried.

7. A method of making an aerobic vaginitis test strip according to claim 3 wherein,

the preparation method of the hydrogen peroxide carrier comprises the following specific steps:

dissolving peroxidase or horseradish peroxidase in a buffer solution with the pH value of 5-8 and the concentration of 0.1-2 mM, adding 0.1-0.6 g/L of a color developing agent and 0.1-1.0 g/L of a reaction promoter to prepare a hydrogen peroxide test solution, wherein the color developing agent is 4-aminoantipyrine, TMB, DHBS, 1, 5-dimethyl-2-phenyl-4-amino-3-pyrazolone (4-AAP); the reaction accelerator is N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-Toluidine (TOOS), 3, 5-dichloro-2-hydroxybenzenesulfonic acid; the buffer solution is one of PBS buffer solution, citric acid-trisodium citrate buffer solution and boric acid-borax buffer solution;

the carrier is soaked in a hydrogen peroxide solution and then dried.

8. A method of making an aerobic vaginitis test strip according to claim 3 wherein,

the specific steps for preparing the pH carrier are as follows:

dissolving bromocresol green and a surfactant in an absolute ethanol solvent to form solution A, wherein the surfactant is one or more of OP emulsifier, triton X-100, tween 20, tween 40, tween 60, tween 80 and polyethylene glycol;

dissolving a stabilizer in purified water or ethanol to form a solution B, wherein the stabilizer is one or more of sodium alginate, trehalose, sodium carboxymethylcellulose, polyvinylpyrrolidone series and gelatin;

mixing the solution A and the solution B according to any ratio of 9: 1-1: 9 to form a pH value solution;

the carrier is soaked in the pH solution and then dried.

Technical Field

The invention relates to the field of in-vitro diagnostic reagents, in particular to an aerobic vaginitis test paper and a preparation method thereof.

Background

The Aerobic Vaginitis (AV) reviewed by the famous gynecologist Donders in belgium, equal to 2011 is characterized by an unbalanced flora and inflammation of the vagina. Clinically, lactobacillus vaginalis is reduced or disappeared and replaced by aerobic bacteria, causing inflammation of vagina to different degrees. Researches show that AV is similar to BV and is easy to cause misdiagnosis, and both the AV and the BV can cause related diseases such as pelvic inflammation, premature birth, newborn low body weight, dead fetus, fetal intrauterine infection, neonatal intelligence disorder and the like in gynecology and obstetrics.

Aerobic vaginitis is the vaginal inflammation resulting from the lack or reduction of hydrogen peroxide-producing lactobacilli by the proliferation of aerobic bacteria, resulting in congestion, edema of the vaginal mucosa and the production of purulent secretions. Common pathogenic bacteria include aerobic bacteria such as group B streptococcus, staphylococcus, Escherichia coli and enterococcus. AV vaginal microecological manifestations: reduction of lactobacilli; the dominant bacteria are diversified and are usually aerobic bacteria such as gram-positive streptococcus, gram-positive coccus, gram-negative coccus or gram-negative bacillus and the like; the pH is often greater than 4.5; hydrogen peroxide (+); (ii) a white blood cell count greater than 10/HP; the Donders score is more than or equal to 3 points; beta-glucuronidase or coagulase is positive.

Most of the methods for AV diagnosis at home and abroad are microscopic method Donders scoring or microorganism culture methods, and the two methods are time-consuming, greatly influenced by subjective factors of operators and inconvenient for clinical routine or inquiry examination. At present, the dry chemical analysis technology is adopted for detection in the market, for example, the invention patent of Beijing Zhongsheng gold Domain, namely a kit for detecting aerobic bacteria in vaginal secretion and a preparation method thereof (patent application No. 200910244267) indicates that beta-glucuronidase and coagulase can effectively diagnose AV. However, the invention only relates to two items of beta-glucuronidase and coagulase, does not relate to leukocyte esterase (leucocyte), hydrogen peroxide (lactobacillus) and pH value, is not comprehensive in detection, and the coagulase method is a two-step method which needs an additional color developing agent, requires incubation for 15min for reaction, is not simple and convenient to operate, has long detection time and needs to be improved.

Disclosure of Invention

The invention aims to provide an aerobic vaginitis test paper and a preparation method thereof, and aims to solve the problems that the conventional test method is incomplete in detection, a color-developing agent is required to be added in a two-step method of a coagulase method, and the reaction is not simple and convenient because incubation is required for 15 min.

In order to achieve the above object, in a first aspect, the present invention provides an aerobic vaginitis test strip, which includes a reaction substrate and a plurality of carriers, wherein after a blank block is reserved on the plurality of carriers, the remaining carriers are prepared by respectively soaking a β -glucuronidase solution, a coagulase solution, a hydrogen peroxide solution, a leukocyte esterase solution and a pH solution, and then drying the carriers, and the plurality of carriers are fixedly disposed on the reaction substrate.

Wherein, the carrier is a test paper block or a reaction hole.

In a second aspect, the present invention provides a method for preparing an aerobic vaginitis test strip, comprising: preparing a beta-glucuronidase carrier; preparing a coagulase carrier; preparing a leukocyte esterase carrier; preparing a hydrogen peroxide carrier; preparing a pH value carrier; all carriers were attached to the reaction substrate, respectively, to complete the preparation.

Wherein the specific steps for preparing the beta-glucuronidase carrier are as follows:

dissolving a substrate in a buffer solution with the pH value of 4-9 and the concentration of 0.5-10 mM, adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction accelerator to form a solution A, wherein the substrate is one or more of 5-bromo-4-chloro-3-indole-beta-D-glucuronide sodium salt, 4-nitrophenyl-beta-D-glucopyranoside (PNPG), 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and phenolphthalein glucuronide, the buffer solution is PBS buffer solution, citric acid-trisodium citrate buffer solution, citric acid-TRIS buffer solution and boric acid-borax buffer solution, one of sodium acetate buffer solution, wherein the reaction promoter is one or more of magnesium chloride, zinc chloride, calcium chloride, glycol, sodium alginate, glutaraldehyde and sucrose; dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5-10 g/L to form a solution B, wherein the diazonium salt color developing agent is one of 2-methoxy-4-morpholinyl benzene diazonium salt, 2-ethoxy-4-morpholinyl benzene diazonium salt and 4-methoxybenzyl diazonium salt; mixing the solution A and the solution B according to a ratio of 1:1 to form a beta-glucuronidase solution; the beta-glucuronidase solution is attached to the carrier and dried.

Wherein the concrete steps for preparing the coagulase carrier are as follows: dissolving a substrate in a buffer solution with the pH value of 3-7 and the concentration of 0.1mM-5mM for buffering, and adding a stabilizer to form a solution A after dissolution is finished, wherein the substrate is glycyl-arginyl-4-methoxy-beta-naphthylamine, the stabilizer is a mixed solution of trehalose and sucrose, and the buffer solution is hydrochloric acid-TRIS buffer solution; dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5-10 g/L to form a B solution, wherein the diazonium salt color developing agent is one of fast violet B salt, fast blue BB salt, fast blue RR salt and fast red B salt; and soaking and drying the carrier in a coagulase A solution, then soaking in a coagulase B solution, and then drying to obtain the carrier.

Wherein, the specific steps for preparing the leucocyte esterase carrier are as follows: dissolving a substrate in a buffer solution with the pH value of 6-9 and the concentration of 0.1-2 mM, adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction promoter to form solution A, wherein the substrate is indoxyl ester and derivatives thereof, one or more of pyrrole ester and pyrrole amino acid ester derivatives thereof, and the stabilizer is one or more of beta-cyclodextrin, mannitol, ethylene glycol, glycerol, sodium ethylene diamine tetraacetate and potassium ethylene diamine tetraacetate; dissolving diazo salt developer in organic solvent containing 0.5-10 g/L organic acid and adding 0.1-5 g/L surfactant to form solution B; mixing the solution A and the solution B according to the proportion of 1: 1-9: 1 to form a leukocyte esterase solution; the carrier is soaked in leukocyte esterase solution and then dried.

Wherein the specific steps for preparing the hydrogen peroxide carrier are as follows: dissolving peroxidase or horseradish peroxidase in a buffer solution with the pH value of 5-8 and the concentration of 0.1-2 mM, adding 0.1-0.6 g/L of a color developing agent and 0.1-1.0 g/L of a reaction promoter to prepare a hydrogen peroxide test solution, wherein the color developing agent is 4-aminoantipyrine, TMB, DHBS, 1, 5-dimethyl-2-phenyl-4-amino-3-pyrazolone (4-AAP); the reaction accelerator is N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-Toluidine (TOOS), 3, 5-dichloro-2-hydroxybenzenesulfonic acid; the buffer solution is one of PBS buffer solution, citric acid-trisodium citrate buffer solution and boric acid-borax buffer solution; the carrier is soaked in a hydrogen peroxide solution and then dried.

Wherein, the specific steps for preparing the pH value carrier are as follows: dissolving bromocresol green and a surfactant in an absolute ethanol solvent to form solution A, wherein the surfactant is one or more of OP emulsifier, triton X-100, tween 20, tween 40, tween 60, tween 80 and polyethylene glycol; dissolving a stabilizer in purified water or ethanol to form a solution B, wherein the stabilizer is one or more of sodium alginate, trehalose, sodium carboxymethylcellulose, polyvinylpyrrolidone series and gelatin; mixing the solution A and the solution B according to any ratio of 9: 1-1: 9 to form a pH value solution; the carrier is soaked in the pH solution and then dried.

The test paper for detecting the aerobic bacterial vaginitis comprises five items of pH value, leukocyte esterase, hydrogen peroxide, beta-glucuronidase and coagulase, and can comprehensively detect and evaluate the aerobic bacterial vaginitis; the one-step method is operated, the color developing solution or the stop solution is not required to be added for detection, the operation is simple, the misoperation is avoided, and the automatic detection is facilitated; the reaction can be detected only by incubating for 5 minutes, the detection time is short, the TAT time is shortened, and the kit is suitable for clinical test detection, particularly hospital outpatient detection screening. The invention relates to an aerobic bacteria vaginitis test paper, wherein the items of beta-glucuronidase and coagulase adopt diazo-azo combination for color development, the reaction color development is obvious, the observation is easy, the test paper is suitable for visual observation, the application scene can be expanded from professional market to personal detection and monitoring, and professional equipment is not needed.

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, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic diagram of an aerobic vaginitis test strip according to the invention;

FIG. 2 is a view showing the construction of an example 1 of the test strip for detecting aerobic vaginitis in accordance with the present invention;

FIG. 3 is a view showing the construction of an aerobic vaginitis test strip of example 2 according to the invention;

FIG. 4 is a flow chart of a method of making an aerobic vaginitis test strip according to the invention.

1-reaction substrate, 2-carrier, 3-test paper block and 4-reaction hole.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

In a first aspect, referring to fig. 1 to 3, the present invention provides an aerobic vaginitis test strip, which includes a reaction substrate 1 and a plurality of carriers 2, wherein the plurality of carriers 2 are prepared by respectively soaking a β -glucuronidase solution, a coagulase solution, a hydrogen peroxide solution, a leukocyte esterase solution, and a pH solution, and drying, and the plurality of carriers 2 are fixedly disposed on the reaction substrate 1.

In the present embodiment, the carrier 2 containing β -glucuronidase comprises a substrate composed of one or more of 5-bromo-4-chloro-3-indole- β -D-glucuronide sodium salt, 4-nitrophenyl- β -D-glucopyranoside (PNPG), 5-bromo-4-chloro-3-indolyl- β -D-glucopyranoside, phenolphthalein glucuronide, 0.1 to 4.0 g/L. 0.1 g/L-4.0 g/L of color developing agent, wherein the color developing agent is a diazonium salt reagent, and the diazonium salt comprises one of 2-methoxy-4-morpholinyl benzene diazonium salt, 2-ethoxy-4-morpholinyl benzene diazonium salt and 4-methoxybenzene diazonium salt. 0.2 g/L-6.0 g/L of reaction accelerator. The solvent is one of PBS buffer solution, citric acid-trisodium citrate buffer solution, citric acid-TRIS buffer solution, boric acid-borax buffer solution and sodium acetate buffer solution. The reaction promoter is one or more of magnesium chloride, zinc chloride, calcium chloride, glycol, sodium alginate, glutaraldehyde and sucrose;

the carrier 2 containing the clotting enzyme test paper block comprises reaction substrate glycyl-arginyl-4-methoxy-beta-naphthylamine with the concentration of 1g/L-10g/L and reaction accelerator diazo salt, wherein the diazo salt is one of fast violet B salt, fast blue BB salt, fast blue RR salt and fast red B salt. The stabilizer is a mixed solution of trehalose and sucrose, and the solvent is hydrochloric acid-TRIS buffer solution;

the carrier 2 containing the leukocyte esterase contains 0.5-1.0 g/L of reaction substrate, 0.1-1.0 g/L of diazonium salt color developing agent, 1.0-10 g/L of sodium chloride, 0.5-1.0 g/L of stabilizer and a buffer solution as a solvent. The reaction substrate is indoxyl ester and derivatives thereof, one or more of pyrrole ester and pyrrole amino acid ester derivatives thereof, and the stabilizer is one or more of beta-cyclodextrin, mannitol, ethylene glycol, glycerol, sodium ethylene diamine tetraacetate and potassium ethylene diamine tetraacetate.

The carrier 2 containing hydrogen peroxide comprises 0.5-1.0 g/L of peroxidase or horseradish peroxidase, 0.1-0.6 g/L of color developing agent, 0.1-1.0 g/L of reaction accelerator and a buffer solution as a solvent. The color developing agent is: 4-aminoantipyrine, TMB, DHBS, 1, 5-dimethyl-2-phenyl-4-amino-3-pyrazolone (4-AAP); the reaction accelerator is N-ethyl-N- (2-hydroxy-3-sulfopropyl) m-Toluidine (TOOS), 3, 5-dichloro-2-hydroxybenzenesulfonic acid; the buffer solution is one of PBS buffer solution, citric acid-trisodium citrate buffer solution and boric acid-borax buffer solution.

The carrier 2 containing the PH value solution comprises 0.1-0.6 g/L of bromocresol green, 0.1-2 g/L of surfactant, 0.1-2 g/L of stabilizer and 10-100% of ethanol in the solution. Wherein the surfactant is one or more of OP emulsifier, Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, and polyethylene glycol. The stabilizer is one or more of sodium alginate, trehalose, sodium carboxymethylcellulose, polyvinylpyrrolidone series, and gelatin.

The invention relates to an aerobic bacterial vaginitis test paper, which comprises five items of PH value, leukocyte esterase, hydrogen peroxide, beta-glucuronidase and coagulase, and can comprehensively detect and evaluate the aerobic bacterial vaginitis; the one-step method is operated, the color developing solution or the stop solution is not required to be added for detection, the operation is simple, the misoperation is avoided, and the automatic detection is facilitated; the reaction can be detected only by incubating for 5 minutes, the detection time is short, the TAT time is shortened, and the kit is suitable for clinical test detection, particularly hospital outpatient detection screening. The invention relates to an aerobic bacteria vaginitis test paper, wherein the items of beta-glucuronidase and coagulase adopt diazo-azo combination for color development, the reaction color development is obvious, the observation is easy, the test paper is suitable for visual observation, the application scene can be expanded from the professional market and monitored by personal detection, and professional equipment is not needed.

Further, the carrier 2 is a test paper block 3 or a reaction hole 4.

In a second aspect, referring to fig. 4, the present invention further provides a method for preparing an aerobic vaginitis test strip, including:

s101, preparing a beta-glucuronidase carrier 2;

s102, preparing a coagulase carrier 2;

s103, preparing a leukocyte esterase carrier 2;

s104, preparing a hydrogen peroxide carrier 2;

s105, preparing a pH value carrier 2;

s106 attaches all carriers 2 to the reaction substrate 1, respectively, completing the preparation.

The following description of two preparation examples is made by dividing the support 2 into the test paper block 3 and the reaction well 4.

Example 1

Dissolving a substrate in a buffer solution with the pH value of 4-9 and the concentration of 0.5-10 mM, and adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction accelerator to form solution A, wherein the substrate consists of one or more of 5-bromo-4-chloro-3-indole-beta-D-glucuronide sodium salt, 4-nitrophenyl-beta-D-glucopyranoside (PNPG), 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and phenolphthalein glucuronide. 0.1 g/L-4.0 g/L of color developing agent, wherein the color developing agent is a diazonium salt reagent, and the diazonium salt comprises one of 2-methoxy-4-morpholinyl benzene diazonium salt, 2-ethoxy-4-morpholinyl benzene diazonium salt and 4-methoxybenzene diazonium salt. 0.2 g/L-6.0 g/L of reaction accelerator. The solvent is one of PBS buffer solution, citric acid-trisodium citrate buffer solution, citric acid-TRIS buffer solution, boric acid-borax buffer solution and sodium acetate buffer solution. The reaction promoter is one or more of magnesium chloride, zinc chloride, calcium chloride, glycol, sodium alginate, glutaraldehyde and sucrose;

and dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5-10 g/L to form a solution B, wherein the color developing agent is a diazonium salt reagent, and the diazonium salt comprises one of 2-methoxy-4-morpholinyl benzene diazonium salt, 2-ethoxy-4-morpholinyl benzene diazonium salt and 4-methoxybenzyl benzene diazonium salt.

Mixing the solution A and the solution B according to a ratio of 1:1 to form a beta-glucuronidase solution;

the test paper block 3 is prepared by drying after being soaked in a beta-glucuronidase solution.

Dissolving a substrate in a buffer solution with the pH value of 3-7 and the concentration of 0.1mM-5mM for buffering, adding a stabilizing agent for dissolution to form solution A, wherein the substrate is glycyl-arginyl-4-methoxy-beta-naphthylamine, and dissolving a diazonium salt color developing agent in an organic acid solution containing 0.5g/L-10g/L to form solution B. And soaking and drying the filter paper in the coagulase A solution, then soaking the filter paper in the coagulase B solution, and then drying the filter paper.

Dissolving the substrate in buffer solution with pH value of 6-9 and concentration of 0.1mM-2mM, and adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction promoter to form solution A. And dissolving the diazonium salt developer in an organic solvent containing 0.5-10 g/L of organic acid, and adding 0.1-5 g/L of surfactant to form solution B. And mixing the solution A and the solution B according to any ratio of 1: 1-9: 1 to form a leukocyte esterase solution. The filter paper was soaked in the leukocyte esterase solution and then dried. The substrate is indoxyl ester and derivatives thereof, one or more of pyrrole ester and pyrrole amino acid ester derivatives thereof, and the stabilizer is one or more of beta-cyclodextrin, mannitol, ethylene glycol, glycerol, sodium ethylene diamine tetraacetate and potassium ethylene diamine tetraacetate.

Dissolving peroxidase or horseradish peroxidase in a buffer solution with the pH value of 5-8 and the concentration of 0.1-2 mM, adding 0.1-0.6 g/L of color developing agent and 0.1-1.0 g/L of reaction accelerator to prepare a hydrogen peroxide test solution. The filter paper was soaked in a hydrogen peroxide solution and then dried.

Dissolving bromocresol green and a surfactant in an absolute ethanol solvent to form solution A. Dissolving the stabilizer in purified water or ethanol to form solution B. And mixing the solution A and the solution B according to any ratio of 9: 1-1: 9 to form a pH value solution. The filter paper is soaked in the pH solution and then dried.

All the test pieces 3 were attached to the reaction substrate 1, respectively, to complete the preparation.

Example 2

Dissolving the substrate in buffer solution with pH value of 4-9 and concentration of 0.5-10 mM, and adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction promoter to form solution A. And dissolving the diazonium salt developer in an organic acid solution containing 0.5-10 g/L to form a solution B. Mixing the solution A and the solution B according to the ratio of 1:1 to form a beta-glucuronidase solution. 5-30ul of beta-glucuronidase solution is dripped into the reaction hole 4 punched by the filter paper, and the beta-glucuronidase reaction hole 4 is formed after drying.

Dissolving the substrate in buffer solution with pH value of 3-7 and concentration of 0.1mM-5mM for buffering, adding stabilizer for dissolution to form solution A, and dissolving diazonium salt developer in organic acid solution containing 0.5g/L-10g/L to form solution B. 5-30ul of coagulase A solution is dripped into the reaction hole 4 punched with filter paper, and then 5-30ul of coagulase A solution is dripped after drying to prepare the reaction hole 4 of coagulase.

Dissolving the substrate in buffer solution with pH value of 6-9 and concentration of 0.1mM-2mM, and adding 1g/L-10g/L stabilizer and 0.5g/L-5g/L reaction promoter to form solution A. And dissolving the diazonium salt developer in an organic solvent containing 0.5-10 g/L of organic acid, and adding 0.1-5 g/L of surfactant to form solution B. And mixing the solution A and the solution B according to any ratio of 1: 1-9: 1 to form a leukocyte esterase solution. 5-30ul of leukocyte esterase solution is dripped into the reaction hole 4 punched with filter paper, and the leukocyte esterase reaction hole 4 is formed after drying.

Dissolving peroxidase or horseradish peroxidase in a buffer solution with the pH value of 5-8 and the concentration of 0.1-2 mM, adding 0.1-0.6 g/L of color developing agent and 0.1-1.0 g/L of reaction accelerator to prepare a hydrogen peroxide test solution. 5-30ul of hydrogen peroxide solution is dripped into the reaction hole 4 punched with the filter paper, and the hydrogen peroxide reaction hole 4 is formed after drying.

Dissolving bromocresol green and a surfactant in an absolute ethanol solvent to form solution A. Dissolving the stabilizer in purified water or ethanol to form solution B. And mixing the solution A and the solution B according to any ratio of 9: 1-1: 9 to form a pH value solution. Dripping 5-30ul of the pH value solution into the reaction hole 4 punched with the filter paper, and drying to form the pH value reaction hole 4.

All the reaction wells 4 are attached to the reaction substrate 1, respectively, to complete the preparation.

The following is a method of using the test strip for detecting aerobic vaginitis of the present invention.

According to the technical scheme, the prepared 5 item test papers are stuck on a substrate and cut into strips to prepare the five-in-one test paper for detecting the aerobic bacteria vaginitis, or 5 reaction holes and 4 combined five-in-one test paper cards for detecting the aerobic bacteria vaginitis.

Rotating the vaginal fornix with a sterile cotton swab or a sterile swab for 10-20 seconds to clearly see the attachment of secretion on the cotton swab or swab. Collecting samples with sterile swabs or cotton swabs, putting the samples into soft test tubes, adding 1.0ml of physiological saline into each sample, and repeatedly washing to fully elute the samples to obtain sample liquid;

taking out the vaginal secretion analysis test paper or the test paper card, and dripping 20-30ul of sample into each item test paper hole, wherein the dripping is about 1 drop;

the test strip is placed on a special instrument for detection, or the test strip is incubated at 37 +/-1 ℃ for 5 minutes, and compared with a color target card within 1 minute to judge the result. The detection results are as follows:

leukocyte esterase:

no color (-) or light purple (+ -) is negative, indicating normal; the mauve (+ 1- +3) is positive, and the color depth indicates that the vagina of the patient has inflammatory reaction with different degrees.

Hydrogen peroxide:

the color is red or purple red as negative (-), which indicates that a large amount of lactobacillus exists possibly and the vaginal flora is normal;

the positive is light red (+), and the weak positive indicates that a medium amount of lactobacillus exists possibly, vaginal bacteria possibly begin to present an abnormal trend or are in a recovery stage, and the negative judgment is usually carried out by combining clinical re-judgment; no color development or yellowish color is positive (+) indicating vaginal dysbacteriosis, vaginal environment morbid or in sub-healthy state.

β -glucuronidase:

no color development is negative (-), indicating normal; the color of red or light purple is weak positive (+), the color of red or purple is positive (+), and the positive or weak positive indicates that the vagina is infected mainly by aerobic bacteria, and the vagina environment is sick or in a sub-health state;

coagulating enzyme:

no color development or pale yellow is negative (-), indicating normal; pale yellow was weakly positive (+); yellow or purple yellow is positive (+), and positive or weak positive indicates that the strain is infected by aerobic bacteria such as staphylococcus aureus, streptococcus and the like;

the clinical significance of the joint examination of each item was as shown in table 1:

TABLE 1

According to national clinical examination operating rules, 600 parts of vaginal secretion samples are tested in total, 52 parts of aerobic bacteria samples are screened out according to a microbiological examination method, and the aerobic bacteria samples in the vaginal secretion are detected by visual comparison of a color standard card, wherein the result of detecting the aerobic bacteria in the vaginal secretion by adopting the kit disclosed by the invention is as follows:

the negative predictive value is: 97.5 percent;

the positive predictive value is: 93.2 percent;

therefore, the detection rate of the detection test paper for the aerobic bacteria vaginitis in the vaginal secretion is more than 90%, the requirements of clinical screening or auxiliary diagnosis can be met, and the detection test paper can be used for monitoring the daily vaginal health condition of women.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.