阅读说明：本技术 一种***炎八联检试剂盒 (Eight joint inspection kit of vaginitis ) 是由 王训琨 路雅丽 娄善梅 于 2020-06-01 设计创作，主要内容包括：本发明公开了一种阴道炎八联检试剂盒,其特征在于,包括反应板,所述反应板具有检测以下指标的反应孔：β-葡萄糖醛酸苷酶、凝固酶、过氧化氢、唾液酸苷酶、白细胞酯酶、乙酰氨基-β-葡萄糖苷酶、脯氨酸氨基肽酶、pH；(2)稀释液；旨在解决无法同时检测需氧菌性阴道炎、细菌性阴道炎、滴虫性阴道炎和霉菌性阴道炎的问题,同时,解决试剂盒检测精度欠缺的问题。(The invention discloses an eight-joint inspection kit for vaginitis, which is characterized by comprising a reaction plate, wherein the reaction plate is provided with reaction holes for detecting the following indexes: beta-glucuronidase, coagulase, hydrogen peroxide, neuraminidase, leukocyte esterase, acetamido-beta-glucosidase, proline aminopeptidase, pH; (2) diluting the solution; the kit aims to solve the problems that aerobic vaginitis, bacterial vaginitis, trichomonas vaginitis and mycotic vaginitis cannot be detected simultaneously and the problem that the kit is lack of detection precision.)

1. The eight-joint vaginitis test kit is characterized by comprising a reaction plate, wherein the reaction plate is provided with reaction holes for detecting the following indexes: beta-glucuronidase, coagulase, hydrogen peroxide, neuraminidase, leukocyte esterase, acetamido-beta-glucosidase, proline aminopeptidase, pH; (2) diluting the solution;

the substrate of the beta-glucuronidase reaction hole of the reaction plate is coated with 5-bromo-4-chloro-3-indole-beta-D-glucuronide and absolute ethyl alcohol;

the substrate of the coagulase reaction hole of the reaction plate is coated with glycyl-arginyl-4-methoxy-beta-naphthamide hydrochloride, trehalose and PBS buffer solution;

the substrate of the hydrogen peroxide reaction hole of the reaction plate is coated with peroxidase, 4-aminoantipyrine, 2,4, 6-tribromo-3-hydroxybenzoic acid, an HRP enzyme stabilizer, sucrose and a PBS buffer solution;

substrates of reaction holes of the reaction plate for neuraminidase are coated with 5-bromo-4-chloro-3-indolyl neuraminic acid salt, absolute ethyl alcohol, distilled water and trehalose;

the substrate of the leukocyte esterase reaction hole of the reaction plate is coated with 5-bromo-4-chloro-3-indoleacetate, trehalose, absolute ethyl alcohol and distilled water;

the substrate of the acetamido-beta-glucosidase reaction hole of the reaction plate is coated with 4-nitrophenyl-N-acetyl-beta-D-glucosaminide, trehalose, absolute ethyl alcohol and distilled water;

the substrate of the proline aminopeptidase reaction hole of the reaction plate is coated with glycylglycine, L-proline paranitroaniline trifluoroacetate, trehalose and PBS buffer salt;

the substrate of the pH reaction hole of the reaction plate is coated with bromocresol green and sodium hydroxide;

the preparation method of the eight-joint inspection kit for vaginitis comprises the following steps:

(1) placing blank filter paper in eight dry chemical reaction holes of a reaction plate, specifically, arranging chamfers in the dry chemical reaction holes, and arranging hole position claws;

(2) respectively dripping corresponding substrate reaction reagent and stabilizer protective solution on blank filter paper of the reaction plate;

(3) and (3) drying: the reaction plate which completed step 2 was dried in an incubator at 37 ℃ for 2 hours.

2. The eight-joint vaginitis test kit according to claim 1, wherein the substrate reaction reagent in the beta-glucuronidase reaction hole is 0.1-5g/L of 5-bromo-4-chloro-3-indoleglucuronide salt in absolute ethyl alcohol, and the stabilizer protection solution in the beta-glucuronidase reaction hole is 20-50g/L of trehalose in aqueous solution.

3. The eight-joint vaginitis test kit according to claim 1, wherein the reaction reagent of the bottom material of the reaction well of the coagulase is glycyl-arginyl-4-methoxy-beta-naphthylamide hydrochloride with the solvent being PBS buffer solution with the concentration of 0.2-4 g/L; the stabilizer protective solution of the coagulase reaction hole is 20-50g/L trehalose aqueous solution.

4. The eight-component vaginitis test kit according to claim 1, wherein the substrate reagents for the hydrogen peroxide reaction wells are: 40-300kU/L of peroxidase, 0.5-2g/L of 4-aminoantipyrine, 0.1-0.5% of 2,4, 6-tribromo-3-hydroxybenzoic acid 5-25g/L, HRP of enzyme stabilizer, PBS buffer solution as solvent, and 0.2-5g/L of sucrose aqueous solution as stabilizer protection solution of hydrogen peroxide reaction hole.

5. The eight-component vaginitis test kit according to claim 1, wherein the substrate reaction reagents of the reaction wells for the neuraminidase are: 0.1-4g/L of 5-bromo-4-chloro-3-indole neuraminic acid salt, and a solvent of absolute ethyl alcohol and distilled water which are mixed according to the volume ratio of 1: 1; the stabilizer protective solution of the reaction hole of the neuraminidase is 20-50g/L aqueous solution of trehalose.

6. The eight-component vaginitis test kit according to claim 1, wherein the substrate reaction reagents of the leukocyte esterase reaction wells are: 0.5-5g/L of 5-bromo-4-chloro-3-indoleacetate, and a solvent of absolute ethyl alcohol and distilled water in a volume ratio of 1: 1; the stabilizer protective solution of the leukocyte esterase reaction hole is 20-50g/L trehalose aqueous solution.

7. The eight-component vaginitis test kit according to claim 1, wherein the substrate reaction reagents of the acetamido-beta-glucosidase reaction hole are as follows: 1-8g/L of 4-nitrophenyl-N-acetyl-beta-D-glucosaminide and a PBS buffer solution as a solvent; the stabilizer protective solution of the acetamido-beta-glucosidase reaction hole is 20-50g/L trehalose aqueous solution.

8. The eight-component vaginitis test kit according to claim 1, wherein said substrate reaction reagent of said proline aminopeptidase reaction well is; 5-20g/L, L g/L glycylglycine-proline p-nitroaniline trifluoroacetate 2-8g/L, a solvent is PBS buffer solution, and a stabilizer protective solution of a proline aminopeptidase reaction hole is 20-50g/L trehalose aqueous solution.

9. The eight-joint vaginitis test kit according to claim 1, wherein the substrate reaction reagent in the pH reaction hole is bromocresol green 0.02-2g/L and NaOH 0.05mol/L, the solvent is water, and the stabilizer protection solution in the pH reaction hole is trehalose aqueous solution 20-50 g/L.

10. The eight-joint inspection kit for vaginitis recited in claim 1, further comprising a color developing agent for developing the colors of reaction wells of neuraminidase and coagulase and a stop solution for reaction wells of acetamido- β -glucosidase, wherein the color developing agent is 0.05% -0.4% of a polyoxyethylene lauryl ether solution of solid violet B; the stop solution is 80g/L sodium hydroxide solution.

Technical Field

The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to an eight-joint inspection kit for vaginitis.

Background

The concept of vaginal microecology is popularized and applied for more than 10 years in China, the evaluation system is improved day by day, and the detection content comprises two aspects of morphology and function. The morphological detection indexes comprise vaginal flora density, diversity, dominant bacteria, pathogenic microorganisms (fungi and trichomonas), Nugent score, white blood cell count and the like. The functional detection indexes comprise pH value, body inflammatory reaction markers (leukocyte esterase) and biochemical indexes, wherein the functional detection indexes comprise detection of metabolites and enzyme activity.

Vaginitis is a common disease in gynecologic outpatients. Common vaginitis includes Bacterial Vaginitis (BV), mycotic vaginitis (VVC), Trichomonas Vaginitis (TV) and Aerobic Vaginitis (AV). Bacterial pre-enzymes are a series of growth metabolism-related enzymes synthesized by bacteria during growth and reproduction, which decompose substrates of various biochemical reactions and retain their activities for a long time in an aerobic environment. The species of the preformed enzyme is different among different species of bacteria. Staphylococcus aureus, enterococcus faecalis and Escherichia coli, which are main pathogenic bacteria of aerobic vaginitis, can produce coagulase, and beta-glucuronidase produced by group B streptococcus and Escherichia coli metabolism; therefore, the coagulase and the beta-glucuronidase can be used for detecting the aerobic bacterial vaginitis. Neuraminidase is a virulence factor of some bacteria, and among bacteria related to bacterial vaginitis, gardnerella, prevotella and the like are known to produce neuraminidase, which is commonly used for detecting bacterial vaginitis. Candida albicans and trichomonas can produce acetamido-beta-glucosidase, and the two can be distinguished by combining pH values.

The vaginitis joint detection kit in the market at present has four-index detection, five-index detection and six-index detection, and can detect bacterial vaginitis, trichomonas vaginitis and colpitis mycotica. However, the kit capable of simultaneously detecting aerobic vaginitis, bacterial vaginitis, trichomonas vaginitis and mycotic vaginitis is rarely reported, more mixed infections exist in clinic, and the joint detection of all indexes is favorable for realizing comprehensive and accurate diagnosis. In addition, the reported kit has the problems of low detection sensitivity and poor stability, and in addition, different reaction wells require respective chromogenic reagents, so the operation is inconvenient.

Disclosure of Invention

The invention aims to provide an eight-joint inspection kit for vaginitis, which aims to solve the problems that aerobic vaginitis, bacterial vaginitis, trichomonas vaginitis and mycotic vaginitis cannot be detected simultaneously and the problem that the detection accuracy of the kit is insufficient.

In order to solve the problems, the technical scheme adopted by the invention is as follows:

an eight-joint vaginitis test kit, which comprises a reaction plate, wherein the reaction plate is provided with reaction holes for detecting the following indexes: beta-glucuronidase, coagulase, hydrogen peroxide, neuraminidase, leukocyte esterase, acetamido-beta-glucosidase, proline aminopeptidase, pH; (2) diluting the solution;

the substrate of the beta-glucuronidase reaction hole of the reaction plate is coated with 5-bromo-4-chloro-3-indole-beta-D-glucuronide, absolute ethyl alcohol, trehalose and distilled water;

the substrate of the coagulase reaction hole of the reaction plate is coated with glycyl-arginyl-4-methoxy-beta-naphthamide hydrochloride, trehalose and PBS buffer solution;

the substrate of the hydrogen peroxide reaction hole of the reaction plate is coated with peroxidase, 4-aminoantipyrine, 2,4, 6-tribromo-3-hydroxybenzoic acid, an HRP enzyme stabilizer, sucrose and a PBS buffer solution;

substrates of reaction holes of the reaction plate for neuraminidase are coated with 5-bromo-4-chloro-3-indolyl neuraminic acid salt, absolute ethyl alcohol, distilled water and trehalose;

the leucocyte esterase substrate of the reaction plate is coated with 5-bromo-4-chloro-3-indoleacetate, trehalose, absolute ethyl alcohol and distilled water;

the acetylamino-beta-glucosidase substrate of the reaction plate is coated with 4-nitrophenyl-N-acetyl-beta-D-glucosaminide, trehalose, absolute ethyl alcohol and distilled water;

the substrate of the proline aminopeptidase reaction hole of the reaction plate is coated with glycylglycine, L-proline paranitroaniline trifluoroacetate, trehalose and PBS buffer salt;

the pH reaction hole of the reaction plate is coated with bromocresol green and sodium hydroxide;

the preparation method of the eight-joint inspection kit for vaginitis comprises the following steps:

(1) placing blank filter paper in eight dry chemical reaction holes of a reaction plate, specifically, arranging chamfers in the dry chemical reaction holes, and arranging hole position claws;

(2) respectively dripping corresponding substrate reaction reagent and stabilizer protective solution on blank filter paper of the reaction plate;

(3) and (3) drying: the reaction plate which completed step 2 was dried in an incubator at 37 ℃ for 2 hours.

The substrate reaction reagent of the beta-glucuronidase reaction hole is 0.1-5 g/L5-bromine-4-chlorine-3-indole glucuronide salt absolute ethyl alcohol solution, and the stabilizer protection solution of the beta-glucuronidase reaction hole is 20-50g/L trehalose aqueous solution.

The reaction reagent of the bottom material of the coagulase reaction hole is glycyl-arginyl-4-methoxyl-beta-naphthamide hydrochloride 0.2-4g/L, and the solvent is PBS buffer solution; the stabilizer protective solution of the coagulase reaction hole is 20-50g/L trehalose aqueous solution.

The substrate reaction reagent of the hydrogen peroxide reaction hole is as follows: 40-300kU/L of peroxidase, 0.5-2g/L of 4-aminoantipyrine, 0.1-0.5% of 2,4, 6-tribromo-3-hydroxybenzoic acid 5-25g/L, HRP of enzyme stabilizer, PBS buffer solution as solvent, and 0.2-5g/L of sucrose aqueous solution as stabilizer protection solution of hydrogen peroxide reaction hole.

The substrate reaction reagent of the reaction hole of the neuraminidase is as follows: 0.1-4g/L of 5-bromo-4-chloro-3-indole neuraminic acid salt, and a solvent of absolute ethyl alcohol and distilled water which are mixed according to the volume ratio of 1: 1; the stabilizer protective solution of the reaction hole of the neuraminidase is 20-50g/L aqueous solution of trehalose.

The substrate reaction reagent of the leukocyte esterase reaction hole is as follows: 0.5-5g/L of 5-bromo-4-chloro-3-indoleacetate, and a solvent of absolute ethyl alcohol and distilled water in a volume ratio of 1: 1; the stabilizer protective solution of the leukocyte esterase reaction hole is 20-50g/L trehalose aqueous solution.

The substrate reaction reagent of the acetamido-beta-glucosidase reaction hole is as follows: 1-8g/L of 4-nitrophenyl-N-acetyl-beta-D-glucosaminide and a PBS buffer solution as a solvent; the stabilizer protective solution of the acetamido-beta-glucosidase reaction hole is 20-50g/L trehalose aqueous solution.

The substrate reaction reagent of the proline aminopeptidase reaction hole is; 5-20g/L, L g/L glycylglycine-proline p-nitroaniline trifluoroacetate 2-8g/L, a solvent is PBS buffer solution, and a stabilizer protective solution of a proline aminopeptidase reaction hole is 20-50g/L trehalose aqueous solution.

The substrate reaction reagent of the pH reaction hole is 0.02-2g/L bromocresol green and 0.05mol/L NaOH, the solvent is water, and the stabilizer protection solution of the pH reaction hole is 20-50g/L trehalose aqueous solution.

The detection kit also comprises a color developing agent for developing reaction holes of the neuraminidase and the coagulase and a stop solution for the reaction holes of the acetamido-beta-glucosidase, wherein the color developing agent is 0.5-4g/L of a polyoxyethylene lauryl ether solution of solid violet B, and the use concentration of the polyoxyethylene lauryl ether is 1-50 g/L; the stop solution is 80g/L sodium hydroxide solution.

The invention has the beneficial effects that:

1. the invention relates to an eight-joint inspection kit for vaginitis, which can directly distinguish bacterial vaginitis, mycotic vaginitis and trichomonas vaginitis by measuring beta-glucuronidase, coagulase, hydrogen peroxide, neuraminidase, leucocyte esterase, acetamido-beta-glucosidase, proline aminopeptidase and pH value, and can further identify the flora such as aerobic bacteria/anaerobic bacteria and facultative anaerobic bacteria in a sample.

2. According to the eight-joint inspection kit for vaginitis, the solvent of a beta-glucuronidase reaction hole is absolute ethyl alcohol, the ethyl alcohol is used as the solvent, the water system component proportion is small, the solubility problem is solved, compared with the method that the ethylene glycol is used as the solvent solution, the eight-joint inspection kit for vaginitis obtains better stability, the ethylene glycol is used as the solvent traditionally, although the dissolving capacity is strong, the metabolic oxidation is easy, the oxalic acid is generated, and the solution stability is poor.

3. According to the invention, a hydrogen peroxide reaction hole substrate is coated with peroxidase, 4-aminoantipyrine, 2,4, 6-tribromo-3-hydroxybenzoic acid, an HRP enzyme stabilizer, sucrose and a PBS buffer solution, when the detection is carried out, hydrogen peroxide in vaginal secretion is acted by peroxidase (HRP) to release nascent oxygen, the nascent oxygen enables the 2,4, 6-tribromo-3-hydroxybenzoic acid to be oxidized to be red or purple in the presence of the 4-aminoantipyrine, the color development depth is in direct proportion to the concentration of the hydrogen peroxide, and compared with the traditional 3, 5-dichloro-2-hydroxy-sodium benzenesulfonate, the sensitivity is greatly improved. The detection principle of the reaction is based on Trinder reaction, hydrogen peroxide is decomposed to generate oxygen under the action of peroxidase, and colorless reduced 4-amino-tipelin and phenol (or derivatives thereof) are coupled, oxidized and condensed to form red quinone compounds. Compared with the commonly used 3, 5-dichloro-2-hydroxybenzoic acid, the 2,4, 6-tribromo-3-hydroxybenzoic acid is easier to react due to the electronic effect of the substituent group, and has higher reaction sensitivity.

4. The eight-joint detection kit for vaginitis, disclosed by the invention, has very wide application of horseradish peroxidase (HRP), but is very unstable and very easy to inactivate at low concentration. The HRP enzyme stabilizer has strong protection effect on the HRP reagent, so that the diluted HRP reagent is stable for more than 12 months at room temperature at extremely low concentration. Generally, the HRP enzyme stabilizer is mainly used for enzyme-labeled antibodies, enzyme-labeled antigens and the like, and the HRP enzyme stabilizer has obvious effect when being used for a reaction device for the first time; experiments show that the storage validity period of the hydrogen peroxide reaction hole is one year at the storage temperature of 2-8 ℃, the activity of HRP can reach 60% after HRP enzyme stabilizer is added and stored for one and half years, and the validity period of the kit can be greatly prolonged by adding the HRP enzyme stabilizer.

5. According to the eight-joint inspection kit for vaginitis, a color development reagent used in reaction holes of a neuraminidase hole is a polyoxyethylene lauryl ether solution of diazonium salt, the polyoxyethylene lauryl ether and the diazonium salt are matched for color development of the neuraminidase hole, and the color development effect is improved by 2 times compared with that of the diazonium salt which is used alone; the polyoxyethylene lauryl ether is easy to dissolve in water, has emulsifying, wetting and dispersing capabilities, is conventionally used as an ether ester nonionic surfactant, is used as an emulsifying wetting agent, is used as a dispersing agent in the rubber industry, and is used as one of components of an oil overflow dispersing agent in the petroleum industry and the environmental protection industry. According to the characteristic of abnormal neuraminidase activity in vaginal secretion of bacterial vaginosis, the neuraminidase hydrolyzes 5-bromo-4-chloro-3-indole neuraminic acid salt to release bromoindolyl, and when encountering diazonium salt and polyoxyethylene lauryl ether, the combination of the diazonium salt and the polyoxyethylene lauryl ether improves the color development sensitivity, the quality control detection sensitivity is 7U/L, and the detection sensitivity of a neuraminidase hole can reach 5.5U/L after the polyoxyethylene lauryl ether is added.

6. According to the eight-joint inspection kit for vaginitis, the neuraminidase hole and the coagulase are simultaneously suitable as a color development liquid, the neuraminidase hydrolyzes 5-bromo-4-chloro-3-indole neuraminic acid salt, and bromoindolyl is released; the coagulase hydrolyzes glycyl-arginyl-4-methoxy-beta-naphthylamide hydrochloride to generate beta-naphthylamine, bromoindole and beta-naphthylamine are aromatic amine substances, the salt B of the fast violet belongs to diazonium salt, the diazonium salt and the aromatic amine are subjected to coupling reaction, polyoxyethylene lauryl ether is a non-ionic surfactant, the addition of the substance can increase the mutual solubility of two phases so as to increase the reaction rate, the color developing agent disclosed by the invention is suitable for two detection holes of neuraminidase and coagulase at the same time, the use of a respective special color developing agent in each traditional reaction hole is changed, the variety of the color developing agent is reduced, and the operation is more convenient.

7. According to the eight-joint inspection kit for vaginitis, a reaction substrate of a pH hole is a combination of bromocresol green and sodium hydroxide, so that the reaction pH value of the hole is closer to the true value of a sample.

Drawings

FIG. 1 is a schematic view of the structure of a reaction plate of the present invention.

FIG. 2 is an enlarged schematic view of the reaction well.

In the figure:

1-reaction plate, 2-reaction hole, 3-filter paper and 4-hole position claw.

Detailed Description

In order to clearly illustrate the technical features of the present solution, the following explains the present solution by way of specific embodiments and with reference to the accompanying drawings.