WO2018001215A1 - 检测细菌性阴道病的试剂盒 - Google Patents

检测细菌性阴道病的试剂盒 Download PDF

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WO2018001215A1
WO2018001215A1 PCT/CN2017/090153 CN2017090153W WO2018001215A1 WO 2018001215 A1 WO2018001215 A1 WO 2018001215A1 CN 2017090153 W CN2017090153 W CN 2017090153W WO 2018001215 A1 WO2018001215 A1 WO 2018001215A1
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solution
reaction
following components
kit
mass percentage
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French (fr)
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周华深
胡守旺
黄若磐
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广州瑞博奥生物科技有限公司
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    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

  • the invention relates to the field of biotechnology, and in particular to a kit for detecting bacterial vaginosis.
  • Bacterial vaginosis is due to a disorder of microecological balance in the vagina, a decrease in normal lactobacilli and Gardnerella vaginalis (GV), Prevotellaspp, Bacteroides, Mobiluncus spp Mycoplasmahominis and other anaerobic bacteria are abnormally increased in number, resulting in a syndrome without vaginal mucosal inflammation.
  • GV lactobacilli and Gardnerella vaginalis
  • Prevotellaspp Bacteroides
  • Mobiluncus spp Mycoplasmahominis and other anaerobic bacteria are abnormally increased in number, resulting in a syndrome without vaginal mucosal inflammation.
  • Haemophilus vaginitis Gardnerella vaginalis vaginitis, non-specific vaginitis and Gardnerella vaginits.
  • they have different names for their pathogens at different times.
  • GV is not the only pathogen causing the disease, there are other anaerobic bacteria and mycoplasma, especially the inflammatory manifestations of this disease are not obvious, the leukocytes in the secretions are scarce, and no pus cells exist. Therefore, It was officially named "Bacterial Vaginosis" at the Swedish International Conference in 1984. About 10% to 50% of patients with BV have no clinical symptoms, mainly relying on laboratory diagnosis.
  • the diagnostic methods of BV are roughly classified into three categories: clinical diagnosis, microbiological diagnosis, and biochemical diagnosis.
  • the clinical diagnosis, the traditional Amsel method is the gold standard for traditional diagnosis of BV. It is simple and easy to perform, but it is susceptible to human factors and BV infection without heads.
  • BV pathogen research it is found that BV is related to certain metabolites in vaginal microorganisms, so BV can be diagnosed by detecting the metabolites of these microorganisms.
  • BV is diagnosed mainly by detecting enzymes related to BV, such as Sialidase.
  • Sialidase also known as neuraminidase, is an enzyme protein found on lysosomes, cytoplasm and cell membranes. This enzyme is very similar to virulence factors and is capable of hydrolyzing non-reducing terminals with various sialic acids. Substrates, such as glycoproteins, glycolipids, gangliosides, and polysaccharides, destroy mucins, enhance bacterial adhesion, and counteract other virulence factors by impairing IgA's immunoreactivity.
  • the sialidase is produced by anaerobic bacteria such as Prevotella and Bacteroides in the vaginal flora, and its activity is considered to be positively correlated with the level of BV infection.
  • BV BLUE kit As BV BLUE kit, it is widely used in many countries in Europe and America because of its simplicity, speed and accuracy. In order to understand the internal environment of the vagina, most of the foreign countries have combined detection of sialidase, hydrogen peroxide, leukocyte esterase and pH.
  • Hydrogen peroxide is an important bactericidal substance produced by Lactobacillus in the vagina. It plays an important role in preventing the increase of pathogenic bacteria and maintaining the balance of vaginal micro-ecology.
  • concentration of hydrogen peroxide in vaginal secretions and lactic acid The number of bacilli is proportional, and the determination of hydrogen peroxide concentration is a good indicator of whether vaginal micro-ecology is normal.
  • concentration of hydrogen peroxide in normal human vaginal secretions is generally 2, such as the detection of hydrogen peroxide concentration ⁇ 2, it means that the micro-ecological bacteria in the vagina have been destroyed, has caused the growth of anaerobic bacteria, leading to the vagina The imbalance of the inner flora.
  • Leukocyte esterase is a protein on the leukocyte membrane.
  • leukocytes especially granulocytes, penetrate into the secretions due to chemotaxis of the epithelial cells.
  • LE is released. Its activity is directly proportional to the number of damaged granulocytes and not necessarily proportional to the number of intact granulocytes.
  • White blood cells and phagocytic cells are the main defensive cells in the vagina.
  • leukocytes are decomposed by hydrolases and endotoxins released by the bacteria they phagocytized, and intracellular LE is released, resulting in increased LE activity in the secretions.
  • LE in vaginal secretions may be normal in trichomonas vaginitis, fungal vaginitis, and BV patients.
  • LE was used as the detection index for the detection of urinary tract infections. Later, it was found that the activity of LE was significantly increased after infection with mycoplasma, chlamydia or gonococcal, and this index was used for the detection and diagnosis of genitourinary infection in women.
  • LE belongs to a wide range of non-specific indicators and is sensitive. Positive indicates that the subject is infected with a pathogen that can synthesize and decompose endotoxin in vaginal cells. Combined with the cleanliness results, the clinic can help the clinician to understand whether the patient's vaginal wall has substantial mucosal damage.
  • vaginal secretions have a pH of 4.0 to 4.5.
  • Lactobacillus in the vagina uses glucone from the vaginal epithelial cells to produce lactic acid and hydrogen peroxide, which keeps the vaginal secretions acidic.
  • the acidic environment is very important for maintaining the vaginal flora, which can prevent the outside world.
  • the invasion of microorganisms and the excessive reproduction of harmful microorganisms are natural barriers to the vagina.
  • the vaginal pH generally rises due to the production of amines by the bacteria and the breakdown of vaginal cells.
  • the routine bacterial vaginosis examination method is microscopic examination.
  • the test items include cleanliness (evaluation of micro-ecological conditions), mold (mold infection index), trichomoniasis (trichomoniasis infection index), pH (acid-base environmental index), amine test. (BV infection index), clue cells (BV infection index).
  • Conventional microscopy has been widely used, but people have strong subjective factors, inconsistent judgment standards, experience accumulation, and high false positive rate.
  • the test items include pH (acid-base environmental indicators), H 2 O 2 (number of beneficial bacteria and bactericidal strength), leukocyte esterase (inflammation index), sialidase (anaerobic) Infection index), proline aminopeptidase (anaerobic and candida infection indicators), acetylglucosaminidase (candida albicans and trichomoniasis infection indicators), uniform test standards, results are intuitive, no experience accumulation, inspection The rate and accuracy are higher than the microscopic examination method, and the sample volume is large and the efficiency is high.
  • Bacterial vaginosis is the most common vaginal non-specific inflammatory disease in obstetrics and gynecology. Its infection rate is about 15% to 50% at home and abroad, and it is often easy to relapse. Its inflammatory response is often recessive. Relatively weak inflammatory response characteristics.
  • the diagnosis of BV mainly uses the clinical diagnostic standard Amsel method, combined with Gram staining, the method is cumbersome to operate, difficult to master, and susceptible to human influence, which is not conducive to timely treatment of patients.
  • Amsel diagnostic criteria 3 of the following 4 items can be diagnosed BV: 1 secretion is a uniform thin leucorrhea; 2 vaginal pH> 4.5; 3 ammonia test positive, take a small amount of vaginal secretions on the glass, 10% oxidized 1 to 2 drops of potassium solution, which produces rotten fish-like odor, which is positive; 4 clue cells are positive, and the clue cells under high power microscope exceed 20%.
  • the kit for the detection of bacterial vaginosis has the characteristics of rapid, simple operation and objective index. It can be used as an indicator for gynecological intravaginal micro-ecological environment evaluation and is used for clinical diagnosis of BV.
  • the dry chemical method for detecting bacterial vaginosis has the following problems: 1.
  • the hydrogen peroxide hole has a high false positive; 2.
  • the room temperature reaction color is weak, and a constant temperature heater is required; 3.
  • Detection index Too much it is easy to confuse in use.
  • the kit for detecting bacterial vaginosis has the characteristics of rapid, simple operation, objective index, and the like, and can be used as a gynecological intravaginal micro-ecological environment. Evaluation indicators for clinically assisted diagnosis of BV.
  • a kit for detecting bacterial vaginosis comprising a detecting device, a color developing solution and a sample diluent, wherein the detecting device comprises a pH reaction pad, a hydrogen peroxide reaction pad, a leukocyte esterase reaction pad and a sialidase Reaction pad
  • the hydrogen peroxide reaction pad is a reaction pad obtained by drying the carrier after being immersed in a hydrogen peroxide reaction solution
  • the hydrogen peroxide reaction solution comprises, by mass percentage, the following components:
  • the sample diluent is expressed in mass percent and includes the following components:
  • the isotonicity adjusting agent is sodium chloride or glucose
  • the mass percentage is 0.5% to 1.1%
  • the mass percentage is 3 % to 6%
  • the pH reaction pad is a reaction pad obtained by drying a carrier after being immersed in a pH reaction solution
  • the pH reaction solution includes a solution A and a sodium hydroxide solution
  • the solution A is mass%.
  • the sialidase reaction pad is a reaction pad obtained by drying a carrier after being immersed in a sialidase reaction solution, and the sialidase reaction solution comprises, by mass percentage, the following components:
  • the sialidase substrate is 5-bromo-4-chloro-3-indolylneuraminic acid, 5-bromo-4-chloro-3-indolyltransferase sodium salt and 4-methylumbelliferone One or more of - ⁇ -N-acetylneuraminic acid sodium salt;
  • the leukocyte esterase reaction pad is a reaction pad obtained by immersing the carrier in a leukocyte esterase reaction solution, and the leukocyte esterase reaction solution comprises, by mass percentage, the following components:
  • Leukocyte esterase substrate 0.001% to 2%
  • the leukocyte esterase substrate is 5-bromo-4-chloro-3-indolyl ethyl ester, 5-bromo-4-chloro-3-indolyl octyl ester, 5-bromo-6-chloro-3-indole
  • 5-bromo-4-chloro-3-indolyl octyl ester 5-bromo-6-chloro-3-indole
  • the color developing solution is in mass percentage and includes the following components:
  • Diazonium salt 0.01% to 5%
  • hydrochloric acid solution a balance of hydrochloric acid solution, acetic acid solution, and biological buffer having a pH of 3.0 to 10.0
  • the diazonium salt is one or more of a solid violet B salt, a solid red B salt, a solid blue B salt, and a 1-diazo-2-naphthol-4-sulfonic acid;
  • the surfactant is One or more of Triton 100, Tween-20, and Tween-80;
  • the ion protectant is magnesium chloride.
  • the solution A in the pH reaction solution comprises the following components:
  • the hydrogen peroxide reaction solution is in mass percent and includes the following components:
  • the sialidase reaction solution is in mass percent and includes the following components:
  • the leukocyte esterase reaction solution is in mass percentage and includes the following components:
  • Leukocyte esterase substrate 0.01% to 1%
  • the color developing solution is in mass percentage and includes the following components:
  • Diazonium salt 0.02% to 2%
  • hydrochloric acid solution a balance of hydrochloric acid solution, acetic acid solution, and biological buffer having a pH of 3.0 to 10.0;
  • the sample diluent is expressed in mass percent and includes the following components:
  • the isotonicity adjusting agent is sodium chloride or glucose, when the isotonicity adjusting agent is sodium chloride, the mass percentage is 0.8% to 1.0%, and when the isotonicity adjusting agent is glucose, the mass percentage is 4.5. % ⁇ 5.5%;
  • the preservative is one or more of sodium azide, polylysine, and ProClin300.
  • the content by mass of the antioxidant in the hydrogen peroxide reaction liquid is more preferably 0.01 to 0.1%, and the content by mass of tetramethylbenzidine is more preferably 0.4% to 0.6%.
  • the mass percentage content of the sialidase substrate in the sialidase reaction solution is more preferably 0.1 to 2%, and the mass percentage content of trehalose is more preferably 1 to 5%.
  • the hydrogen peroxide reaction solution comprises, by mass percent, 5% to 10% of sodium 3,5-dichloro-2-hydroxybenzenesulfonate, and 0.04% to 0.1% of 4-amino group.
  • Antipyrine 0.01% to 1% horseradish peroxidase, 0.07% to 0.08% antioxidant, the balance being biological buffer.
  • the hydrogen peroxide reaction liquid is preferably preferably 7-8% of sodium 3,5-dichloro-2-hydroxybenzenesulfonate, 0.04 to 0.06% of 4-aminoantipyrine, 0.4% by mass. ⁇ 0.6% horseradish peroxidase, 0.07-0.08% antioxidant, the balance is biological buffer.
  • the sample diluent is in mass percent and includes the following components:
  • the solution A in the pH reaction solution comprises the following components:
  • Alizarin red S 0.005% to 0.01%
  • the sialidase reaction solution comprises, by mass percent, 0.1% to 1% of 5-bromo-4-chloro-3-indolyl sodium salt, 2% to 5% of seaweed Sugar, 0.5% to 1% sodium sulfate, the balance is biological buffer.
  • the sialidase reaction solution is preferably preferably 0.1 to 0.2% 5-bromine in terms of mass percentage -4-Chloro-3-purine neuraminic acid sodium salt, 2 to 3% trehalose, 0.5 to 0.6% sodium sulfate, the balance being biological buffer.
  • the leukocyte esterase reaction solution comprises, by mass percentage, 0.09% to 1% of 5-bromo-4-chloro-3-indolyl ethyl ester, 0.5% to 1% of imidazole, and the balance is Anhydrous ethanol.
  • the leukocyte esterase reaction solution further preferably comprises 0.9 to 1% of 5-bromo-4-chloro-3-indolyl ethyl ester, 0.5 to 0.6% of imidazole, and the balance is anhydrous ethanol.
  • the color developing solution comprises, by mass percentage, 0.02% to 1% solid purple B salt, 0.08% to 0.1% Triton 100, 0.4% to 5% magnesium chloride, and the balance is pH. It is a biological buffer of 3.0 to 10.0.
  • the color developing solution is preferably a mass buffer including 0.1 to 0.2% solid purple B salt, 0.08 to 0.1% triton 100, 4 to 5% magnesium chloride, and the balance being a biological buffer having a pH of 3.0 to 10.0. liquid.
  • the biological buffer is one or more of a phosphate buffer, an acetate buffer, a citrate buffer, and a TRIS buffer, and the concentration of the biological buffer is 0.01 mol/L. ⁇ 2 mol/L; the antioxidant is one or more of vitamin C, polyphenol and tocopherol.
  • the carrier in the present invention is one of a glass cellulose film, a nitrocellulose membrane, absorbent paper, filter paper, and chromatographic filter paper.
  • the carrier is preferably a filter paper or an absorbent paper.
  • the present invention is directed to the deficiencies of the prior art.
  • By improving the formulation of the hydrogen peroxide reaction solution and the sample diluent it is possible to improve the high false positive of the hydrogen peroxide pores and the reaction at room temperature, and further, by improving the pH reaction liquid, the white blood cell ester.
  • the formulation of the enzyme reaction solution, the sialidase reaction solution, and the color developing solution enables the kit to react at room temperature and the test result is intuitive and accurate; the kit for detecting bacterial vaginosis of the present invention has a room temperature condition
  • the characteristics of the next reaction, simple operation, and objective indicators can be used as indicators for gynecological intravaginal microecological environment evaluation for clinical diagnosis of BV.
  • 1 is a comparison diagram of detection effects of a pH reaction liquid in an embodiment of the present invention
  • FIG. 3 is a comparison diagram of detection effects of a sialidase reaction solution in an embodiment of the present invention.
  • Fig. 5 is a comparison diagram of the detection effect of the color developing liquid in the embodiment of the present invention.
  • the raw materials used in the present invention are all commercially available ordinary raw materials.
  • the invention relates to a kit for detecting bacterial vaginosis, which comprises a kit body, a detecting device, a color developing solution and a sample diluent.
  • the detection device comprises four reaction pads, which are a pH reaction pad, a hydrogen peroxide reaction pad, a leukocyte esterase reaction pad, a sialidase reaction pad, and the carriers of the four reaction pads are 3MM filter paper, reagent a pH reaction well, a hydrogen peroxide reaction well, a leukocyte esterase reaction well and a sialidase reaction well are respectively arranged on the box, and each reaction well is filled with a corresponding reaction pad;
  • the pH reaction pad is a reaction pad obtained by immersing the carrier in a pH reaction solution, and the specific formula of the pH reaction solution is as follows:
  • the rest is anhydrous ethanol
  • the solution A was adjusted to a green to blue color with a sodium hydroxide solution.
  • the rest is anhydrous ethanol
  • the solution A was adjusted to a green to blue color with a sodium hydroxide solution.
  • Solution A bromocresol green: 0.2 wt%
  • Alizarin red S 0.01wt%
  • the rest is anhydrous ethanol
  • the solution A was adjusted to a green to blue color with a sodium hydroxide solution.
  • Fig. 1 the results obtained from recipes 1, 2, and 3 from left to right in Fig. 1). It can be seen from Fig. 1 that the reaction pads treated by the formula of the present invention have distinct color changes and good effects, and wherein the formula 3 is The treated reaction pad achieves the best detection results.
  • the hydrogen peroxide reaction pad is a reaction pad obtained by immersing the carrier in a hydrogen peroxide reaction solution, and the hydrogen peroxide reaction solution comprises, by mass percentage, the following components:
  • Vitamin C 0.075%
  • the rest was 0.2 mol/L acetate buffer.
  • Vitamin C 0.075%
  • the rest is anhydrous ethanol.
  • the test results of the above two formulations of the hydrogen peroxide reaction solution were compared.
  • the specific steps are as follows: 35 ⁇ L of the reaction pad after the treatment of the above two formulations of hydrogen peroxide reaction solution were respectively added at a concentration of 0 ⁇ mol/L (pure Water), 2 ⁇ mol/L, 4 ⁇ mol/L, 8 ⁇ mol/L, 16 ⁇ mol/L, 32 ⁇ mol/L, 64 ⁇ mol/L, 128 ⁇ mol/L hydrogen peroxide solution (the above hydrogen peroxide solution is used in a concentration of 30% hydrogen peroxide solution)
  • the sample dilution of the present invention is diluted.
  • the specific formulation of the sample diluent is 5wt% gum arabic, 0.001wt% ProClin300, 0.9wt% sodium chloride, and the hydrochloric acid is added to adjust the pH of the sample diluent to 3.5, and the balance is The purified water was reacted at room temperature (10 ° C to 30 ° C) for 30 minutes, and the results were observed.
  • Figure 2 Figure 2-A is the result of Formulation 1
  • Figure 2-B is the result of Formulation 2.
  • the sample diluent of the invention can make the hydrogen peroxide more stable, prevent the hydrogen peroxide in the sample from decomposing during the inspection or detection process, thereby reducing the problem of high false positive hydrogen peroxide.
  • sialidase reaction solution is expressed by mass percent, and includes the following components:
  • Trehalose 5%
  • the rest was 0.2 mol/L acetate buffer.
  • the rest was 0.2 mol/L phosphate buffer.
  • Trehalose 1%
  • reaction mats prepared by the above four formulations of the sialidase reaction solution were subjected to a comparison test, and the specific steps were as follows: a concentration of 35 ⁇ L was added to the reaction pad after the treatment of the above four formulations of the sialidase reaction solution.
  • sialidase solution (the above sialidase solution is diluted with the sample dilution of the present invention, and the specific formulation of the sample diluent is 5wt % gum arabic, 0.001 wt% ProClin 300, 0.9 wt% sodium chloride, hydrochloric acid added to adjust the pH of the sample dilution to 3.5, the balance is purified water) and the color developing solution of the invention (specific formula is 0.2% solid) Purple B salt, 0.1% Triton 100, 5% magnesium chloride, the balance is hydrochloric acid solution of pH 3.0)
  • the test was carried out, and the reaction was carried out for 30 minutes at room temperature (10 ° C to 30 ° C), and the results were observed.
  • Figure 3 Figure 3-A for the results of Formulation 1, Figure 3-B for the results of Formulation 2, Figure 3-C for the results of Formulation 3, Figure 3-D for the results of Formulation 4
  • Figure 3 It can be seen that the reaction pad treated by the formula of the invention is sensitive, and the initial concentration can be detected as 0.00375 U/mL, and the color change of each concentration is distinct, and the effect is good, and the reaction pad adopting formula 2 has the best effect, and the best detection effect can be achieved. .
  • the leukocyte esterase reaction pad is a reaction pad obtained by immersing the carrier in a leukocyte esterase reaction solution, and the leukocyte esterase reaction solution comprises, by mass percentage, the following components:
  • the rest is anhydrous ethanol.
  • the rest is anhydrous ethanol.
  • the rest is anhydrous ethanol.
  • the rest is anhydrous ethanol.
  • Oxime ester 0.1%
  • the rest is anhydrous ethanol.
  • the rest is anhydrous ethanol.
  • the rest is anhydrous ethanol.
  • the comparison results of the above eight formulas of leukocyte esterase reaction solution were as follows: the concentration of 35 ⁇ L was 0.1094 U/mL in the reaction pad after the treatment of the above eight formulas of leukocyte esterase reaction solution, 0.2188 U/mL, 0.4375 U/mL, 0.875 U/mL, 1.75 U/mL leukocyte esterase solution (the above leukocyte esterase solution is diluted with the sample diluent of the present invention, and the specific formulation of the sample diluent is 5 wt% gum arabic, 0.001wt% ProClin300, 0.9wt% sodium chloride, the amount of hydrochloric acid added is the pH of the sample dilution to 3.5, the balance is purified water) and the color developing solution of the invention (the specific formula is 0.2% solid purple B salt, 0.1% Triton 100, 5% magnesium chloride, and the balance is a hydrochloric acid solution of pH 3.0.) The test was carried out at room temperature (10 °
  • Figure 4 Figure 4-A is the result of Formulation 1, Figure 4-B is the result of Formulation 2, Figure 4-C is the result of Formulation 3, Figure 4-D is the result of Formulation 4, Figure 4-E For the results of Formulation 5, Figure 4-F is the result of Formulation 6, Figure 4-G is the result of Formulation 7, and Figure 4-H is the result of Formulation 8.
  • Figure 4 It can be seen from Figure 4 that the reaction pad treated by the formulation of the present invention is sensitive. , the initial concentration can be detected to be 0.1094 U/mL, and each concentration The color changes are distinct and the effect is good, and the reaction pad using the formula 1 has the best effect, and the best detection effect can be achieved.
  • hydrochloric acid solution having a pH of 3.0.
  • Solid red B salt 0.02%
  • hydrochloric acid solution having a pH of 3.0.
  • Solid blue B salt 0.02%
  • the reaction pad was prepared by taking the leukocyte esterase reaction solution of the formula 1 of the present invention, and adding 35 ⁇ L of the concentration of 0.1094 U/mL, 0.2188 U/mL, 0.4375 U/mL, 0.875 U/mL, and 1.75 U/mL leukocyte esterase, respectively. At the same time, the color development liquid of the above four formulations was added, and the reaction was carried out for 30 minutes at room temperature (10 ° C to 30 ° C), and the results were observed.
  • the color developing solution of the invention is used for a sialidase reaction pad and a leukocyte esterase reaction pad, and the above two reaction pads can respectively use different color developing liquids or the same color developing liquid.
  • the pH reaction pad is prepared by immersing the carrier in a pH reaction solution and drying it in an environment of 10 ° C to 50 ° C to obtain a completely dry pH reaction pad;
  • the hydrogen peroxide reaction pad is obtained by immersing the carrier through the hydrogen peroxide reaction solution, and drying it in an environment of 10 ° C to 50 ° C to obtain a completely dried hydrogen peroxide reaction pad;
  • the leukocyte esterase reaction pad is obtained by immersing the carrier through the leukocyte esterase reaction solution, and drying it in an environment of 10 ° C to 50 ° C to obtain a completely dried leukocyte esterase reaction pad;
  • the sialidase reaction pad is obtained by immersing the carrier in a sialidase reaction solution, and drying it in an environment of 10 ° C to 50 ° C to obtain a completely dried sialidase reaction pad;
  • a pH reaction well a hydrogen peroxide reaction well, a leukocyte esterase reaction well, and a sialidase reaction well are disposed on the reagent cartridge body, and each reaction well is filled with a corresponding reaction pad, that is, Got it.
  • the kit principle of the invention for detecting bacterial vaginosis is the kit principle of the invention for detecting bacterial vaginosis
  • leukocyte esterase activity used to indicate the catalytic activity of leukocyte esterase in the sample solution, suggesting whether the subject has vaginosis.
  • the leukocyte esterase in the sample solution can hydrolyze the leukocyte esterase substrate to release the bromine thiol group.
  • the diazonium salt is reversed, it is red, yellow, purple or blue, showing color depth and white blood cells.
  • the activity of esterase is directly proportional.
  • sialidase activity used to indicate the catalytic activity of sialidase, suggesting whether the subject has bacterial vaginosis.
  • the sialidase hydrolyzes the sialidase substrate to release the bromo thiol group.
  • the reaction is red, yellow, magenta or blue in response to the diazonium salt, and the degree of coloration is proportional to the sialidase activity.
  • pH reaction pad The normal vaginal pH is generally 4.0-4.5, and the pH reaction pad detection range is 3.6-5.4. As the pH value increases, the pH reaction pad color transitions from yellow to blue.
  • a drop (about 35 ⁇ l) of a sample is added to the leukocyte reaction well, the sialidase well, and the hydrogen peroxide reaction well on the kit of the present invention, and then in the leukocyte esterase reaction well and the sialidase reaction well. Add one drop of the coloring solution to each drop.
  • the kit of the present invention is reacted at room temperature (10 ° C to 30 ° C) for 30 minutes, and the result is immediately judged, or placed in a water bath or a dry bath at a constant temperature of 37 ° C, and the bath is warmed for 15 minutes, and the result is immediately judged.
  • Hydrogen peroxide reaction well negative in red or purplish red or blue, indicating that the vaginal flora may be normal, no discoloration is positive, indicating that the vaginal flora may be out of tune;
  • Leukocyte esterase reaction well no discoloration is negative; blue-green, blue or red is positive, indicating varying degrees of inflammatory response in the vagina.
  • pH reaction well pH value of the reaction well yellow or yellow-green indicates that the sample pH ⁇ 4.5, the pH value of the reaction well is blue-green, blue indicates the sample pH> 4.5;
  • the kit for detecting bacterial vaginosis according to the invention adopts 100 effective samples of the top three hospitals for detection, and the gold standard method (microscopic examination) confirms that the positive cases of bacterial vaginosis are 41 cases, and the kit for detecting bacterial vaginosis of the present invention is confirmed by the kit. 42 cases of positive cases of bacterial vaginosis, the coincidence rate of 85%; gold standard method The negative case of bacterial vaginosis was 59 cases.
  • the kit for detecting bacterial vaginosis of the present invention confirmed 58 cases of negative cases of bacterial vaginosis, the coincidence rate was 88%; the kit for detecting bacterial vaginosis of the present invention and hospital gold
  • the results of the standard method are compared as follows
  • kit for detecting bacterial vaginosis by the present invention is highly consistent with the clinical microscopic examination effect.

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Abstract

本发明公开了一种检测细菌性阴道病的试剂盒，包括检测装置、显色液和样本稀释液，所述检测装置中包括pH值反应垫、过氧化氢反应垫、白细胞酯酶反应垫和唾液酸苷酶反应垫。

Description

检测细菌性阴道病的试剂盒 技术领域
本发明涉及生物技术领域，特别是涉及一种检测细菌性阴道病的试剂盒。
背景技术
细菌性阴道病(bacterial vaginosis，BV)是由于阴道内微生态平衡失调，正常乳酸杆菌减少而阴道加特纳菌(Gardnerella vaginalis,GV)、普雷沃菌(Prevotellaspp)、拟杆菌(Bacteroides)、动弯杆菌(Mobiluncus spp)人型支原体(Mycoplasmahominis)等兼性厌氧菌数量异常增加，所引起的一种无阴道粘膜炎症表现的综合症。以前对它的认识有限，曾命名为嗜血杆菌阴道炎(Haemophilus vaginitis)、棒状杆菌阴道炎(Gardnerella vaginalis vaginitis)、非特异性阴道炎(NO-specific vaginitis)和加特纳菌阴道炎(Gardnerella vaginits)等，皆因在不同时期对其病原体认识不同而命名不同。1955年Gardner和Dukes从特异性阴道炎患者的阴道分泌物中分离出阴道嗜血杆菌，认为是本病的病原菌，因而称此病为阴道嗜血杆菌阴道炎(Corynebacterium vaginitis)。1980年Greenwood和Pickett通过DNA-DNA杂交试验，电镜观察及细菌外膜生化分析证实此菌是一独立新菌种，并命名为阴道加特纳菌(GV)，改称此病为加特纳菌阴道炎。但由于GV并不是导致该病的唯一病原体，还有其他厌氧菌和支原体等存在，特别是此病的炎性表现并不明显，分泌物中白细胞稀少，也无脓细胞存在，因此，在1984年的瑞典国际会议上被正式命名为“细菌性阴道病”。BV患者约10％～50％无临床症状，主要依靠实验室诊断。BV的诊断方法大致分为三类即临床诊断、微生物学诊断、生物化学诊断。临床诊断即传统的Amsel法，是传统诊断BV的金标准，简单易行，但易受人为因素及BV感染无头的因素影响，主观性强，操作费时，已逐渐被其他方法所取代。微生物学检查操作繁琐，效率低，已很少使用。随着对BV病原学研究的深入，发现BV与阴道微生物中的某些代谢产物有关，因此可以通过检测这些微生物的代谢产物来诊断BV，目前主要通过检测与BV相关的酶来诊断BV，如唾液酸苷酶。
唾液酸酶又称神经氨酸酶，是一种存在于溶酶体、细胞质和细胞膜上的酶蛋白，该酶活性与毒力因子很相似，能够水解非还原性的终端为唾液酸的多种底物，如糖蛋白、糖脂类、神经节苷脂以及多聚糖类，破坏粘蛋白，增强细菌黏附性，而且通过损害IgA的免疫反应性来对抗其他毒力因子。唾液酸苷酶由阴道菌群中的厌氧菌如普雷沃菌和拟杆菌等产生，其活性被认为与BV感染水平呈正相关。唾液酸苷酶的活性在BV时(1.35±1.87nmol/ml min)明显高于无BV的妇女(0.03±0.14nmol/ml min)该法的检测原理是，人工合成唾液酸酶底物，并以其酶与底物反应后显示颜色的不同来诊断BV。
近年来利用致病菌特异的酶催化机理，结合发色底物形成的酶快速检测技术，已成为BV快速诊断发展的主导方向。如BV BLUE试剂盒，以其简便、快速、准确的优点在欧美许多国家广泛应用。为了对阴道内环境做进一点的了解，目前国外大多将唾液酸苷酶、过氧化氢、白细胞酯酶、pH值等几项进行联合检测。
过氧化氢是阴道内乳酸杆菌产生的一种重要的杀菌物质，对防止致病菌的加剧增值和维持阴道微生态平衡具有重要的作用，一般情况下，阴道分泌物中过氧化氢浓度与乳酸杆菌的数量成正比，过氧化氢浓度的测定是分析阴道微生态是否正常的良好指标。正常人阴道分泌物中的过氧化氢浓度一般在2，如检测过氧化氢浓度≤2时，则意味着阴道内微生态菌遭到到破坏，已经引起了厌氧菌的生长，导致了阴道内菌群的失衡。
白细胞酯酶(Leukocyte esterase，LE)是白细胞膜上的一种蛋白质，炎症感染时白细胞特别是粒细胞由于趋化作用通道上皮细胞粘膜渗透到分泌物中，当粒细胞受到破坏以后释放出LE，其活性与受损的粒细胞数量成正比，而与完整的粒细胞数量不一定成比例。白细胞和吞噬细胞是阴道中主要的防御性细胞。细菌感染时，白细胞被其吞噬的细菌释放的水解酶和内毒素分解，胞内的LE释放出来，造成分泌物中LE活性增高。其他微生物如滴虫、念珠菌和加特纳菌等对白细胞的毒性较低，因此滴虫性阴道炎、霉菌性阴道炎和BV患者阴道分泌物中的LE可能正常。LE作为检测指标最早用于尿道感染的检测，后来人们发现支原体、衣原体或淋球菌感染时LE的活性明显升高后，才把此指标用于女性的泌尿生殖感染的检测诊断。LE属于广泛的非特异性指标，较敏感，阳性提示受检者阴道内感染了能合成分解阴道细胞内毒素的病原体，结合清洁度结果，可以帮助临床医生了解患者阴道壁有无实质性粘膜损伤。
正常阴道分泌物pH值为4.0～4.5，阴道内的乳酸杆菌利用阴道上皮细胞的糖原产生乳酸和过氧化氢，使阴道分泌物保持酸性，酸性环境对维持阴道菌群非常重要，可阻止外界微生物的入侵和有害微生物的过度繁殖，是阴道的天然屏障。患者有BV时，由于细菌自产和分解阴道细胞产生的胺类化合物，阴道pH普遍上升。
常规的细菌性阴道病检查方法为镜检，检测项目包括清洁度(评估微生态状况)，霉菌(霉菌感染指标)，滴虫(滴虫感染指标)，pH(酸碱环境指标)，胺试验(BV感染指标)，线索细胞(BV感染指标)。常规镜检已经普及使用，但人为主观因素强，判断标准不一致，需要经验积累，误判率高。作为阴道病检查的干化学酶法，检测项目包括pH(酸碱环境指标)，H2O2(有益菌数量及杀菌强弱)，白细胞酯酶(炎症指标)，唾液酸苷酶(厌氧菌感染指标)，脯氨酸氨基肽酶(厌氧菌及念珠菌感染指标)，乙酰氨基葡萄糖苷酶(白色念珠菌及滴虫感染指标)，检测标准统一，结果直观，无需经验积累，检出率和准确性高于镜检法，且标本量大，效率高。
细菌性阴道病是妇产科最常见的一种阴道非特异性炎性疾病，国内外报道其感染率约为15％～50％，且常易复发，它的炎症反应常常是隐性的，具有比较弱的炎症反应特点。
目前，诊断BV主要采用临床诊断标准Amsel法，同时结合革兰氏染色，该方法操作繁琐，且不易掌握，易受人为影响，不利于患者及时治疗.
Amsel诊断标准：以下4项中符合3项即可诊断BV：①分泌物为均匀一致的稀薄白带；②阴道pH>4.5；③氨试验阳性,取少量阴道分泌物于玻璃片上,10％氢氧化钾溶液1～2滴,产生烂鱼样腥臭味即为阳性；④线索细胞阳性,高倍显微镜下线索细胞超过20％。
细菌性阴道病联合检测试剂盒，具有快速、操作简单、指标客观等特点，可作为妇科阴道内微生态环境评价的指标，用于临床辅助诊断BV。
对比市面的存在的三个公司的干化学法检测细菌性阴道病试剂盒，存在以下问题：1、过氧化氢孔假阳性高；2、室温反应颜色弱，需要恒温加热器；3、检测指标过多，使用中容易混淆。
发明内容
基于此，有必要针对上述问题，提供一种检测细菌性阴道病的试剂盒，所述检测细菌性阴道病的试剂盒具有快速、操作简单、指标客观等特点，可作为妇科阴道内微生态环境评价的指标，用于临床辅助诊断BV。
为实现上述技术目的，具体技术方案如下：
一种检测细菌性阴道病的试剂盒，包括检测装置、显色液和样本稀释液，所述检测装置中包括pH值反应垫、过氧化氢反应垫、白细胞酯酶反应垫和唾液酸苷酶反应垫；
其中，所述过氧化氢反应垫为载体经过氧化氢反应液浸泡后干燥所得的反应垫，所述过氧化氢反应液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000001
所述样本稀释液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000002
所述等渗调节剂为氯化钠或葡萄糖，当所述等渗调节剂为氯化钠时，质量百分比为0.5％～1.1％，当所述等渗调节剂为葡萄糖时，质量百分比为3％～6％。
在其中一些实施例中，所述pH值反应垫为载体经pH值反应液浸泡后干燥所得的反应垫，所述pH值反应液包括溶液A和氢氧化钠溶液，所述溶液A以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000003
Figure PCTCN2017090153-appb-000004
用氢氧化钠溶液调节所述溶液A颜色为绿色至蓝色；
所述唾液酸苷酶反应垫为载体经唾液酸苷酶反应液浸泡后干燥所得的反应垫，所述唾液酸苷酶反应液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000005
所述唾液酸苷酶底物为5-溴-4-氯-3-吲哚神经氨酸、5-溴-4-氯-3-吲哚神经氨酸钠盐和4-甲基伞形酮-α-N-乙酰基神经氨酸苷钠盐中的一种或一种以上；
所述白细胞酯酶反应垫为载体经白细胞酯酶反应液浸泡后干燥所得的反应垫，所述白细胞酯酶反应液以质量百分比计，包括以下组分：
白细胞酯酶底物    0.001％～2％，
咪唑              0％～5％，
无水乙醇          余量；
所述白细胞酯酶底物为5-溴-4-氯-3-吲哚乙酯、5-溴-4-氯-3-吲哚辛酯、5-溴-6-氯-3-吲哚乙酯、5-溴-6-氯-3-吲哚辛酯、吲哚酯、吡咯酯和2-甲氧基-4-吗啉基重氮苯氯化锌盐中的一种或一种以上；
所述显色液以质量百分比计，包括以下组分：
重氮盐          0.01％～5％，
表面活性剂      0.001％～1％，
离子保护剂      0.01％～10％，
pH值为3.0～10.0的盐酸溶液、醋酸溶液、生物缓冲液中的一种余量，
所述重氮盐为固紫B盐、固红B盐、固蓝B盐和1-重氮基-2-萘酚-4-磺酸中一种或一种以上；所述表面活性剂为曲拉通100、吐温-20和吐温-80中的一种或一种以上；所述离子保护剂为氯化镁。
在其中一些实施例中，所述pH值反应液中的溶液A包括以下组分：
Figure PCTCN2017090153-appb-000006
Figure PCTCN2017090153-appb-000007
所述过氧化氢反应液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000008
所述唾液酸苷酶反应液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000009
所述白细胞酯酶反应液以质量百分比计，包括以下组分：
白细胞酯酶底物    0.01％～1％，
咪唑              0％～2％，
无水乙醇          余量；
所述显色液以质量百分比计，包括以下组分：
重氮盐          0.02％～2％，
表面活性剂      0.01％～0.1％，
离子保护剂      0.1％～5％，
pH值为3.0～10.0的盐酸溶液、醋酸溶液、生物缓冲液中的一种余量；
所述样本稀释液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000010
Figure PCTCN2017090153-appb-000011
所述等渗调节剂为氯化钠或葡萄糖，当所述等渗调节剂为氯化钠时，质量百分比为0.8％～1.0％，当所述等渗调节剂为葡萄糖时，质量百分比为4.5％～5.5％；
所述防腐剂为叠氮钠、多聚赖氨酸、ProClin300中的一种或一种以上。
所述过氧化氢反应液中的抗氧化剂的质量百分比含量进一步优选为0.01～0.1％，四甲基联苯胺的质量百分比含量进一步优选为0.4％～0.6％。
所述唾液酸苷酶反应液中的唾液酸苷酶底物的质量百分比含量进一步优选为0.1～2％，海藻糖的质量百分比含量进一步优选为1～5％。
在其中一些实施例中，所述过氧化氢反应液以质量百分比计，包括5％～10％的3,5-二氯-2-羟基苯磺酸钠，0.04％～0.1％的4-氨基安替比林，0.01％～1％的辣根过氧化物酶，0.07％～0.08％的抗氧化剂，余量为生物缓冲液。
所述过氧化氢反应液以质量百分比计，进一步优选为包括7～8％的3,5-二氯-2-羟基苯磺酸钠，0.04～0.06％的4-氨基安替比林，0.4～0.6％的辣根过氧化物酶，0.07～0.08％的抗氧化剂，余量为生物缓冲液。
在其中一些实施例中，所述样本稀释液以质量百分比计，包括以下组分：
Figure PCTCN2017090153-appb-000012
在其中一些实施例中，所述pH值反应液中的溶液A包括以下组分：
溴甲酚绿  0.1％～0.2％，
茜素红S   0.005％～0.01％，
无水乙醇  余量。
在其中一些实施例中，所述唾液酸苷酶反应液以质量百分比计，包括0.1％～1％5-溴-4-氯-3-吲哚神经氨酸钠盐，2％～5％海藻糖，0.5％～1％硫酸钠，余量为生物缓冲液。
所述唾液酸苷酶反应液以质量百分比计，进一步优选为包括0.1～0.2％5-溴 -4-氯-3-吲哚神经氨酸钠盐，2～3％海藻糖，0.5～0.6％硫酸钠，余量为生物缓冲液。
在其中一些实施例中，所述白细胞酯酶反应液以质量百分比计，包括0.09％～1％5-溴-4-氯-3-吲哚乙酯，0.5％～1％咪唑，余量为无水乙醇。
所述白细胞酯酶反应液以质量百分比计，进一步优选为包括0.9～1％5-溴-4-氯-3-吲哚乙酯，0.5～0.6％咪唑，余量为无水乙醇。
在其中一些实施例中，所述显色液以质量百分比计，包括0.02％～1％固紫B盐，0.08％～0.1％曲拉通100，0.4％～5％氯化镁，余量为pH值为3.0～10.0的生物缓冲液。
所述显色液以质量百分比计，进一步优选为包括0.1～0.2％固紫B盐，0.08～0.1％曲拉通100，4～5％氯化镁，余量为pH值为3.0～10.0的生物缓冲液。
在其中一些实施例中，所述生物缓冲液为磷酸缓冲液、醋酸缓冲液、柠檬酸缓冲液和TRIS缓冲液中的一种或一种以上，所述生物缓冲液的浓度为0.01mol/L～2mol/L；所述抗氧化剂为维生素C、多酚和生育酚中的一种或一种以上。
本发明中所述载体为玻璃纤维素膜、硝酸纤维素膜、吸水纸、滤纸和层析滤纸中的一种。上述载体优选滤纸、吸水纸。
本发明相较现有技术的优点以及有益效果为：
本发明针对现在技术的不足，通过改进过氧化氢反应液和样本稀释液的配方，可以改善过氧化氢孔假阳性高以及在室温条件下反应，进一步的，通过改进pH值反应液、白细胞酯酶反应液、唾液酸苷酶反应液、显色液的配方，使本试剂盒在室温条件下也能反应且检查结果直观准确；本发明检测细菌性阴道病的试剂盒，具有可在室温条件下反应、操作简单、指标客观等特点，可作为妇科阴道内微生态环境评价的指标，用于临床辅助诊断BV。
附图说明
图1为本发明实施例中的pH值反应液的检测效果对比图；
图2为本发明实施例中的过氧化氢反应液的检测效果对比图；
图3为本发明实施例中的唾液酸苷酶反应液的检测效果对比图；
图4为本发明实施例中的白细胞酯酶反应液的检测效果对比图；
图5为本发明实施例中的显色液的检测效果对比图。
具体实施方式
以下通过实施例进一步说明本发明。
本发明中所用原料均为市售普通原料。
实施例
一种检测细菌性阴道病的试剂盒，该试剂盒由试剂盒体，检测装置、显色液和样本稀释液组成。检测装置中包括四种反应垫，分别是pH值反应垫、过氧化氢反应垫、白细胞酯酶反应垫、唾液酸苷酶反应垫，所述的四种反应垫的载体均为3MM滤纸，试剂盒体上分别设置有pH值反应孔、过氧化氢反应孔、白细胞酯酶反应孔以及唾液酸苷酶反应孔，每个反应孔中填入对应的反应垫；
(1)pH值反应垫为载体经pH值反应液浸泡后干燥所得的反应垫，pH值反应液的具体配方如下：
配方1：
溶液A：溴甲酚绿：0.1wt％，
其余为无水乙醇；
用氢氧化钠溶液调上述溶液A颜色为绿色至蓝色。
配方2：
溶液A：溴甲酚绿：0.02wt％，
溴酚蓝：0.01wt％，
其余为无水乙醇；
用氢氧化钠溶液调上述溶液A颜色为绿色至蓝色。
配方3：
溶液A：溴甲酚绿：0.2wt％；
茜素红S：0.01wt％；
其余为无水乙醇；
用氢氧化钠溶液调上述溶液A颜色为绿色至蓝色。
对以上三个配方的pH值反应液所处理的反应垫进行了检测结果对比试验，具体步骤如下：
向以上三种配方pH值反应液的处理的反应垫中加入35μL pH值分别为3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4的醋酸缓冲液，立即观察结果。
结果请见图1(图1中从左至右分别为配方1、2、3所得结果)，从图1可知本发明配方处理的反应垫，各个浓度颜色变化分明，效果好，而其中配方3处理的反应垫可达到最佳的检测效果。
(2)过氧化氢反应垫为将载体经过氧化氢反应液浸泡后干燥所得的反应垫，所述过氧化氢反应液以质量百分比计，包括以下组分：
配方1：
3,5-二氯-2-羟基苯磺酸钠：7.5％；
4-氨基安替比林：0.05％；
辣根过氧化物酶：0.5％；
维生素C：0.075％；
其余为0.2mol/L的醋酸缓冲液。
配方2：
四甲基联苯胺：0.5％；
辣根过氧化物酶：0.5％；
维生素C：0.075％；
其余为无水乙醇。
对以上两个配方的过氧化氢反应液进行了检测结果对比试验，具体步骤如下：向在以上两种配方过氧化氢反应液处理后的反应垫中加入35μL的浓度分别为0μmol/L(纯水)、2μmol/L、4μmol/L、8μmol/L、16μmol/L、32μmol/L、64μmol/L、128μmol/L过氧化氢溶液(上述过氧化氢溶液使用浓度为30％的过氧化氢溶液采取本发明样本稀释液进行稀释，样本稀释液的具体配方为5wt％阿拉伯树胶，0.001wt％ProClin300，0.9wt％氯化钠，盐酸添加量为调样本稀释液的pH值至3.5，余量为纯化水)，在室温(10℃～30℃)中，反应30分种，观察结果。
结果请见图2(图2-A为配方1的结果，图2-B为配方2的结果)，从图2可知本发明配方处理的反应垫反应灵敏，可检测初始浓度为2μmol/L，且各个浓度颜色变化分明，效果好，而其中采用配方1的反应垫效果最好，可达到最佳的检测效果。本发明样本稀释液可使过氧化氢更为稳定，防止样本中的过氧化氢在送检或检测过程中分解，从而减少了过氧化氢假阳性高的问题。
(3)唾液酸苷酶反应垫为载体经唾液酸苷酶反应液浸泡后干燥所得的反应 垫，所述唾液酸苷酶反应液以质量百分比计，包括以下组分：
配方1：
5-溴-4-氯-3-吲哚神经氨酸：1％；
海藻糖：5％；
硫酸钠：1％；
其余为0.2mol/L的醋酸缓冲液。
配方2：
5-溴-4-氯-3-吲哚神经氨酸钠盐：0.1％；
海藻糖：2.5％；
硫酸钠：0.52％；
其余为0.2mol/L的磷酸缓冲液。
配方3：
4-甲基伞形酮-α-N-乙酰基神经氨酸苷钠盐：0.1％；
海藻糖：2.5％；
硫酸钠：0.52％；
其余为0.2mol/L的柠檬酸缓冲液。
配方4：
5-溴-4-氯-3-吲哚神经氨酸：0.01％；
5-溴-4-氯-3-吲哚神经氨酸钠盐：2％；
4-甲基伞形酮-α-N-乙酰基神经氨酸苷钠盐：2％；
海藻糖：1％；
硫酸钠：1％；
其余为0.2mol/L TRIS缓冲液。
对以上四个配方的唾液酸苷酶反应液所制备的反应垫进行了检测结果对比试验，具体步骤如下：向在以上4种配方唾液酸苷酶反应液处理后的反应垫中加入35μL的浓度分别为0.00375U/mL、0.0075U/mL、0.015U/mL、0.03U/mL唾液酸苷酶溶液(上述唾液酸苷酶溶液采取本发明样本稀释液进行稀释，样本稀释液的具体配方为5wt％阿拉伯树胶，0.001wt％ProClin300，0.9wt％氯化钠，盐酸添加量为调样本稀释液的pH值至3.5，余量为纯化水)以及本发明的显色液(具体配方为0.2％固紫B盐，0.1％曲拉通100，5％氯化镁，余量为pH3.0的盐酸溶液) 进行检测，在室温(10℃～30℃)中，反应30分种，观察结果。从配方1和配方4的结果可以看到0.03U/mL的唾液酸苷酶浓度已经达到了设计要求，结果与0.06U/mL相差不大，因此在配方2和配方3中设置最高检测浓度为0.03U/ML，不再设置0.06U/mL浓度。
结果请见图3(图3-A为配方1的结果，图3-B为配方2的结果，图3-C为配方3的结果，图3-D为配方4的结果)，从图3可知本发明配方处理的反应垫反应灵敏，可检测初始浓度为0.00375U/mL，且各个浓度颜色变化分明，效果好，而其中采用配方2的反应垫效果最好，可达到最佳的检测效果。
(4)所述白细胞酯酶反应垫为载体经白细胞酯酶反应液浸泡后干燥所得的反应垫，所述白细胞酯酶反应液以质量百分比计，包括以下组分：
配方1：
5-溴-4-氯-3-吲哚乙酯：1％；
咪唑：0.5％；
其余为无水乙醇。
配方2：
5-溴-4-氯-3-吲哚辛酯：1％；
咪唑：5％；
其余为无水乙醇。
配方3：
5-溴-6-氯-3-吲哚乙酯：0.5％；
咪唑：0；
其余为无水乙醇。
配方4：
5-溴-6-氯-3-吲哚辛酯：0.1％；
咪唑：1％；
其余为无水乙醇。
配方5：
吲哚酯：0.1％；
咪唑：0.1％；
其余为无水乙醇。
配方6：
吡咯酯：1％；
咪唑：5％；
其余为无水乙醇。
配方7：
2-甲氧基-4-吗啉基重氮苯氯化锌盐：1％；
咪唑：5％；
其余为无水乙醇。
配方8：
5-溴-4-氯-3-吲哚乙酯：0.2％
5-溴-4-氯-3-吲哚辛酯：0.2％
5-溴-6-氯-3-吲哚乙酯：0.2％
5-溴-6-氯-3-吲哚辛酯：0.2％
吲哚酯：0.2％
吡咯酯：0.2％
2-甲氧基-4-吗啉基重氮苯氯化锌盐：0.2％
咪唑：2.5％
其余为无水乙醇
对以上8个配方的白细胞酯酶反应液进行了检测结果对比试验，具体步骤如下：向在以上8种配方白细胞酯酶反应液处理后的反应垫中加入35μL的浓度分别为0.1094U/mL、0.2188U/mL、0.4375U/mL、0.875U/mL、1.75U/mL白细胞酯酶溶液(上述白细胞酯酶溶液采取本发明样本稀释液进行稀释，样本稀释液的具体配方为5wt％阿拉伯树胶，0.001wt％ProClin300，0.9wt％氯化钠，盐酸添加量为调样本稀释液的pH值至3.5，余量为纯化水)以及本发明的显色液(具体配方为0.2％固紫B盐，0.1％曲拉通100，5％氯化镁，余量为pH3.0的盐酸溶液。)进行检测，在室温(10℃～30℃)中，反应30分种，观察结果。
结果请见图4(图4-A为配方1的结果，图4-B为配方2的结果，图4-C为配方3的结果，图4-D为配方4的结果，图4-E为配方5的结果，图4-F为配方6的结果，图4-G为配方7的结果，图4-H为配方8的结果)，从图4可知本发明配方处理的反应垫反应灵敏，可检测初始浓度为0.1094U/mL，且各个浓度 颜色变化分明，效果好，而其中采用配方1的反应垫效果最好，可达到最佳的检测效果。
(5)显色液以质量百分比计，具体配方如下：
配方1：
固紫B盐：0.2％；
曲拉通100：0.1％；
氯化镁：5％；
pH值为3.0的盐酸溶液余量。
配方2：
固红B盐：0.02％；
曲拉通100：0.01％；
氯化镁：0.1％；
pH值为3.0的盐酸溶液余量。
配方3：
固蓝B盐：0.02％；
曲拉通100：0.01％；
氯化镁：0.1％；
pH值为9.0的0.2mol/L TRIS缓冲液余量。
配方4：
1-重氮基-2-萘酚-4-磺酸：0.01％；
曲拉通100：0.01％；
氯化镁：1％；
pH值为7.0的0.2mol/L TRIS缓冲液余量。
对以上四个配方的显色液进行了检测结果对比试验，具体步骤如下：
取本发明配方1的白细胞酯酶反应液制备反应垫，加入35μL的浓度分别为0.1094U/mL、0.2188U/mL、0.4375U/mL、0.875U/mL、1.75U/mL白细胞酯酶，并同时加入上述4个配方的显色液行检测，在室温(10℃～30℃)中，反应30分种，观察结果。
结果请见图5(图5-A为配方1的结果，图5-B为配方2的结果，图5-C为配方3的结果，图5-D为配方4的结果)，从图5可知，本发明配方的显色液各 个浓度颜色变化分明，效果好，其中配方1效果最理想，能达到最佳的检测效果。
本发明显色液用于唾液酸苷酶反应垫以及白细胞酯酶反应垫，上述两反应垫可分别使用不同配方的显色液或同一种配方的显色液。
上述检测细菌性阴道病的试剂盒的制备方法：
反应垫的制备：
pH值反应垫为将载体经pH值反应液浸泡后，在10℃～50℃的环境下进行干燥处理得到完全干燥的pH值反应垫；
过氧化氢反应垫为将载体经过氧化氢反应液经浸泡后，在10℃～50℃的环境下进行干燥处理得到完全干燥的过氧化氢反应垫；
白细胞酯酶反应垫为将载体经白细胞酯酶反应液经浸泡后，在10℃～50℃的环境下进行干燥处理得到完全干燥的白细胞酯酶反应垫；
唾液酸苷酶反应垫为将载体经唾液酸苷酶反应液经浸泡后，在10℃～50℃的环境下进行干燥处理得到完全干燥的唾液酸苷酶反应垫；
按照本发明配方配置样本稀释液和显色液；
采用常规制备试剂盒体，在试剂盒体上设置pH值反应孔、过氧化氢反应孔、白细胞酯酶反应孔以及唾液酸苷酶反应孔，每个反应孔中填入对应的反应垫，即得。
本发明检测细菌性阴道病的试剂盒原理：
1、过氧化氢浓度测定：用于表示分泌物中有益菌如乳酸杆菌的多少，阴性表明乳酸杆菌正常，阳性则表明乳酸杆菌偏少或没有，提示阴道菌群失调，受检者可能处于病变或亚健康状态。在样本液中的过氧化氢存在条件下，耦联色原底物4-氨基安替吡啉、DHBS(3,5-二氯-2-羟基苯磺酸钠)和TMB(四甲基联苯胺)在HRP(辣根过氧化物酶)的作用下缩合形成红色、紫红色或蓝色的物质，且呈色深浅与过氧化氢的浓度成正比。
2、白细胞酯酶活性测定：用于表示样本液中的白细胞酯酶的催化活性，提示受检者是否有阴道病。样本液中的白细胞酯酶能崔化水解白细胞酯酶底物，释放出溴吲哚基，遇重氮盐起反呈红色、黄色、紫红色或蓝色，呈色深浅与白细胞 酯酶的活性成正比。
3、唾液酸苷酶活性测定：用于表示唾液酸苷酶的催化活性，提示受检者是否有细菌性阴道病。唾液酸苷酶水解唾液酸苷酶底物，释放出溴吲哚基，遇重氮盐起反应呈红色、黄色、紫红色或蓝色，呈色程度与唾液酸苷酶活性成正比。
4、pH值反应垫：正常阴道pH一般为4.0-4.5，pH反应垫的检测范围是3.6-5.4，随着pH值的升高，pH值反应垫颜色从黄色到蓝色过渡。
本发明检测细菌性阴道病的试剂盒操作流程
将取样棉签取样后先在本发明试剂盒上的pH值反应孔中点印一下，立即观察结果；然后将取样棉签放入试管中，加入400微升样本稀释液于试管中，反复挤压管壁，使目的物充分释出。
在本发明试剂盒上的白细胞酶反应孔、唾液酸苷酶孔、过氧化氢反应孔滴加一滴(约35微升)的样品，然后在白细胞酯酶反应孔和唾液酸苷酶反应孔中各滴加一滴显色液。
把本发明试剂盒在室温(10℃～30℃)中，反应30分种，立即判断结果，或置于恒温37℃的水浴或干浴中，温浴15分钟，立即判断结果。
结果判断：
过氧化氢反应孔：呈红色或紫红色或蓝色为阴性，指示阴道菌群可能正常，不变色为阳性，指示阴道菌群可能失调；
白细胞酯酶反应孔：不变色为阴性；蓝绿色、蓝色或红色为阳性，指示阴道内有不同程度的炎症反应。
唾液酸苷酶反应孔：不变色为阴性；蓝绿色、蓝色或红色为阳性，指示可能感染细菌性阴道病；
pH值反应孔：pH值反应孔显黄色或黄绿色表示样品pH≤4.5，pH值反应孔显蓝绿色、蓝色表明样品pH＞4.5；
临床对比实验
采用本发明检测细菌性阴道病的试剂盒采用三甲医院100例有效样本进行检测，金标准法(镜检)确认细菌性阴道病阳性病例为41例，本发明检测细菌性阴道病的试剂盒确认细菌性阴道病阳性病例42例，符合率达85％；金标准法确 认细菌性阴道病阴性病例为59例，本发明检测细菌性阴道病的试剂盒确认细菌性阴道病阴性病例58例，符合率达88％；本发明检测细菌性阴道病的试剂盒与医院金标准法的结果对比如下
Figure PCTCN2017090153-appb-000013
可见，采用本发明检测细菌性阴道病的试剂盒与临床镜检效果高度一致。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种检测细菌性阴道病的试剂盒，其特征在于，包括检测装置、显色液和样本稀释液，所述检测装置中包括pH值反应垫、过氧化氢反应垫、白细胞酯酶反应垫和唾液酸苷酶反应垫；
    其中，所述过氧化氢反应垫为载体经过氧化氢反应液浸泡后干燥所得的反应垫，所述过氧化氢反应液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100001
    所述样本稀释液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100002
    所述等渗调节剂为氯化钠或葡萄糖，当所述等渗调节剂为氯化钠时，质量百分比为0.5％～1.1％，当所述等渗调节剂为葡萄糖时，质量百分比为3％～6％。
  2. 根据权利要求1所述的检测细菌性阴道病的试剂盒，其特征在于，所述pH值反应垫为载体经pH值反应液浸泡后干燥所得的反应垫，所述pH值反应液包括溶液A和氢氧化钠溶液，所述溶液A以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100003
    Figure PCTCN2017090153-appb-100004
    用氢氧化钠溶液调节所述溶液A颜色为绿色至蓝色；
    所述唾液酸苷酶反应垫为载体经唾液酸苷酶反应液浸泡后干燥所得的反应垫，所述唾液酸苷酶反应液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100005
    所述唾液酸苷酶底物为5-溴-4-氯-3-吲哚神经氨酸、5-溴-4-氯-3-吲哚神经氨酸钠盐和4-甲基伞形酮-α-N-乙酰基神经氨酸苷钠盐中的一种或一种以上；
    所述白细胞酯酶反应垫为载体经白细胞酯酶反应液浸泡后干燥所得的反应垫，所述白细胞酯酶反应液以质量百分比计，包括以下组分：
    白细胞酯酶底物  0.001％～2％，
    咪唑            0％～5％，
    无水乙醇    余量；
    所述白细胞酯酶底物为5-溴-4-氯-3-吲哚乙酯、5-溴-4-氯-3-吲哚辛酯、5-溴-6-氯-3-吲哚乙酯、5-溴-6-氯-3-吲哚辛酯、吲哚酯、吡咯酯和2-甲氧基-4-吗啉基重氮苯氯化锌盐中的一种或一种以上；
    所述显色液以质量百分比计，包括以下组分：
    重氮盐      0.01％～5％，
    表面活性剂  0.001％～1％，
    离子保护剂  0.01％～10％，
    pH值为3.0～10.0的盐酸溶液、醋酸溶液、生物缓冲液中的一种余量，
    所述重氮盐为固紫B盐、固红B盐、固蓝B盐和1-重氮基-2-萘酚-4-磺酸中一种或一种以上；所述表面活性剂为曲拉通100、吐温-20和吐温-80中的一种或一种以上；所述离子保护剂为氯化镁。
  3. 根据权利要求2所述的检测细菌性阴道病的试剂盒，其特征在于，所述pH值反应液中的溶液A包括以下组分：
    Figure PCTCN2017090153-appb-100006
    Figure PCTCN2017090153-appb-100007
    所述过氧化氢反应液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100008
    所述唾液酸苷酶反应液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100009
    所述白细胞酯酶反应液以质量百分比计，包括以下组分：
    白细胞酯酶底物  0.01％～1％，
    咪唑            0％～2％，
    无水乙醇  余量；
    所述显色液以质量百分比计，包括以下组分：
    重氮盐      0.02％～2％，
    表面活性剂  0.01％～0.1％，
    离子保护剂  0.1％～5％，
    pH值为3.0～10.0的盐酸溶液、醋酸溶液、生物缓冲液中的一种余量；
    所述样本稀释液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100010
    Figure PCTCN2017090153-appb-100011
    所述等渗调节剂为氯化钠或葡萄糖，当所述等渗调节剂为氯化钠时，质量百分比为0.8％～1.0％，当所述等渗调节剂为葡萄糖时，质量百分比为4.5％～5.5％；
    所述防腐剂为叠氮钠、多聚赖氨酸、ProClin300中的一种或一种以上。
  4. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述过氧化氢反应液以质量百分比计，包括5％～10％的3,5-二氯-2-羟基苯磺酸钠，0.04％～0.1％的4-氨基安替比林，0.01％～1％的辣根过氧化物酶，0.07％～0.08％的抗氧化剂，余量为生物缓冲液。
  5. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述样本稀释液以质量百分比计，包括以下组分：
    Figure PCTCN2017090153-appb-100012
  6. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述pH值反应液中的溶液A包括以下组分：
    溴甲酚绿  0.1％～0.2％，
    茜素红S   0.005％～0.01％，
    无水乙醇  余量。
  7. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述唾液酸苷酶反应液以质量百分比计，包括0.1％～1％ 5-溴-4-氯-3-吲哚神经氨酸钠盐，2％～5％海藻糖，0.5％～1％硫酸钠，余量为生物缓冲液。
  8. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述白细胞酯酶反应液以质量百分比计，包括0.09％～1％ 5-溴-4-氯-3-吲哚乙酯，0.5％～1％咪唑，余量为无水乙醇。
  9. 根据权利要求3所述的检测细菌性阴道病的试剂盒，其特征在于，所述显色液以质量百分比计，包括0.02％～1％固紫B盐，0.08％～0.1％曲拉通100，0.4％～5％ 氯化镁，余量为pH值为3.0～10.0的生物缓冲液。
  10. 根据权利要求1～9任一项所述的检测细菌性阴道病的试剂盒，其特征在于，所述生物缓冲液为磷酸缓冲液、醋酸缓冲液、柠檬酸缓冲液和TRIS缓冲液中的一种或一种以上，所述生物缓冲液的浓度为0.01mol/L～2mol/L；所述抗氧化剂为维生素C、多酚和生育酚中的一种或一种以上。
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