WO2010102529A1 - 滴虫和念珠菌的检测 - Google Patents
滴虫和念珠菌的检测 Download PDFInfo
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- WO2010102529A1 WO2010102529A1 PCT/CN2010/070382 CN2010070382W WO2010102529A1 WO 2010102529 A1 WO2010102529 A1 WO 2010102529A1 CN 2010070382 W CN2010070382 W CN 2010070382W WO 2010102529 A1 WO2010102529 A1 WO 2010102529A1
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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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
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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
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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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/39—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts
- G01N2333/40—Assays involving biological materials from specific organisms or of a specific nature from fungi from yeasts from Candida
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
Definitions
- the present invention relates to a method and kit for detecting the presence or absence of Trichom onas in a specimen by detecting N-acetyl- ⁇ -D-hexosaminidase activity.
- the invention also relates to methods and kits for the differential detection of the presence of trichomoniasis or Candida (0» ⁇ 3 ⁇ 4 ⁇ ) in a specimen.
- the present invention relates to a method and kit for the combined detection of vaginal diseases caused by trichomoniasis, candida, bacteria or other causes. current technology
- vaginalis Three species of trichomoniasis are known to be parasitic in the human body: vaginalis, parasitic in the vagina, urethra and prostate, causing trichomonas vaginitis, etc.; Trichomonas vaginalis (2: hominis), parasitic in the intestine , causing intestinal trichomoniasis; Trichomonas vaginalis (tewox), parasitic in the mouth, tartar and frog holes, causing oral trichomoniasis.
- the methods for checking trichomoniasis mainly include: hanging drop method, observing the motility of trichomoniasis, but it is greatly affected by temperature; smear staining method, smearing the specimen after treatment or without treatment, after staining, microscopic examination, this The method is interfered by particles similar in size to the trichomoniasis, and is affected by the field of view; the culture method, after targeted amplification of the trichomoniasis in the specimen, and then smear staining, thus taking a long time; immunological method, its use Specific antibodies detect trichomoniasis antigens, such as fluorescent antibody assays, ELISA methods, latex agglutination methods, etc., but high-quality immunological products for clinical trichomoniasis detection have not been found so far.
- Candida in the human body is mostly a conditional pathogen, which can be found in the oral cavity and genitourinary tract. It is currently known that more than 85 % of the conditional pathogenic Candida species are Candida albicans (C. i//b o»w) and C. tropicalis, which cause urogenital diseases in humans.
- the methods for inspecting Candida mainly include: direct smear microscopy, women use sterile cotton swabs to take vaginal, cervical secretions or milky white film on the vaginal wall, males scrape the scales of the glans glans, coronal sulcus or foreskin surface lesions
- Direct smear microscopy Sub- or looking for hyphae, this method depends on experience, and is affected by the visual field, interference particles, especially the paraffin oil added by the specimen lubrication; fungal culture method, using a special medium such as Sabouraud agar , Example 3 ⁇ 4 mouth Oxoid Ltd., Basingstoke, UK) targeted amplification of Candida in the specimen, which takes a long time; latex agglutination test, polyclonal antibody reacts with mannan of Candida, but no High quality products that can be used in clinical practice.
- kits such as, for example, the kits described below:
- a kit comprising N-acetyl- ⁇ -D-hexosaminoglycoside as a reagent for detecting trichomoniasis.
- the kit of item 1.1 also includes a pH indicator.
- N-acetyl- ⁇ -D-hexosinoside is N-acetyl- ⁇ -D-glucosaminide or N-acetyl- ⁇ -D-galactoside.
- the N-acetyl- ⁇ -D-hexosinoside has a chromogen, preferably the chromogen is bromoindolyl, p-nitrophenyl, 4-indolyl umbelliferone Or naphthol group.
- kit of any of the above further comprising at least one reagent selected from the group consisting of: a reagent for detecting sialidase, a reagent for detecting proline aminopeptidase, and a peroxidase reagent for detecting hydrogen peroxide. , and reagents for detecting leukocyte esterase, and the like.
- kit of any of the above further comprising a standard colorimetric card, such as a NAG standard color chart and/or a pH standard color chart.
- the reagent for detecting leukocyte esterase, and at least one reagent for pH indicator are solidified on a solid phase carrier, preferably the solid phase carrier is selected from paper and plastic having adsorption capacity,
- the paper is, for example, cotton pulp paper, wood pulp paper, sugar cane pulp paper and glass fiber filter paper.
- kit of any of the above which is a joint check card, comprising a card base and a sealing body disposed on the card base.
- the kit of item 1.9 wherein the sealing body is a sealing cover that is fastened to a side of the slot of the reaction zone of the card base.
- N-acetyl- ⁇ -D-hexosinoside is N-acetyl- ⁇ -D-glucosaminide or N-acetyl- ⁇ -D-galactoside.
- the N-acetyl- ⁇ -D-hexosinoside carries a chromogen, preferably the chromogen is bromoindolyl, p-nitrophenyl, 4-indolyl umbelliferone or Naphthol group.
- N-acetyl- ⁇ -D-hexosinoside is cured on a solid support
- the solid support is selected from paper and plastic having an adsorption capacity, such as cotton. Pulp paper, wood pulp paper, sugar cane pulp paper and fiberglass filter paper.
- a further aspect of the invention relates to a detection method, in particular for example the method described in the following:
- a method of detecting the presence of trichomoniasis in a specimen comprising contacting N-acetyl- ⁇ -D-aminohexosides with a specimen.
- Methods for the differential detection of the presence of trichomoniasis and Candida in a specimen including contacting N-acetyl- ⁇ -D-hexosaminoglycoside with a specimen, and also contacting the pH indicator with the specimen.
- N-acetyl- ⁇ -D-hexosinoside has a chromogen, preferably the chromogen is bromoindolyl, p-nitrophenyl, 4-indolyl umbelliferone or Naphthol group.
- the N-acetyl- ⁇ -D-hexosinoside is cured on a solid support
- the solid support is selected from the group consisting of paper and plastic having an adsorption capacity, such as cotton pulp. Paper, wood pulp paper, sugar cane pulp paper and fiberglass filter paper.
- N-acetyl- ⁇ -D-hexosinoside is judged as No Trichomonas or Candida, or N-acetyl- ⁇ -D-hexosinoside is positive and H value No less than 4.8 was judged to have trichomoniasis, or N-acetyl- ⁇ -D-hexosinoside was positively reacted and the pH was not higher than 4.6 and it was judged to have Candida.
- Combined detection method comprising performing the method of any of the above, and performing at least one test selected from the group consisting of: sialidase detection, proline aminopeptidase detection, hydrogen peroxide detection, and leukocyte esterase Detection, etc.
- N-acetyl- ⁇ -D-hexosaminidase means that it is classified as EC.3.2.1.52 according to the nomenclature of the Nomenclature Committee of the International Union of Biochemistry. Enzyme. It mainly acts on N-acetyl- ⁇ -D-glucosamine substrate and N-acetyl- ⁇ -D-galactoside substrate, hydrolyzing the terminal non-reducing N of N-acetyl- ⁇ -D-hexosinoside -acetyl-D-ammonia Hexose.
- This enzyme is an inducing enzyme, and an analog of the recognition group to which the enzyme is added can induce secretion of the enzyme.
- the enzyme-inducing agent refers to a substance capable of inducing secretion of the N-acetyl- ⁇ -D-hexosaminidase of the present invention, and may be selected from galactose, glucose, fructose and the like.
- aliases such as hexosaminidase; ⁇ -acetylaminodeoxyhexosidase; ⁇ -acetyl- ⁇ -D-hexosaminidase (N- ace tyl-) P-hexosaminidase; N-acetyl-beta-hexosaminidase; ⁇ -hexosaminidase; ⁇ -acetylhexosaminidase ⁇ -DN-acetylhexosaminidase; ⁇ -N-acetylglucosaminidase; ⁇ -N-acetyl-D-hexosaminidase ( ⁇ -) N-acetyl-D-hexosaminidase); hexosaminidase A; N-acetylhexosaminidase; ⁇ -D-hexosaminidase, they All are included in the scope of
- Compounds which are substrates for the N-acetyl- ⁇ -D-hexosaminidase of the present invention are also known as, for example, ⁇ -acetylhexosinoside; N-acetyl- ⁇ -D-hexosinoside; N-acetyl-beta -aminohexosides; ⁇ -aminohexosides; ⁇ -DN-acetylhexosinoside; ⁇ -N-acetylglucosaminide; ⁇ -N-acetyl-D-hexosinoside; ⁇ -acetamidohexose; or ⁇ a compound of -D-hexosinoside or the like.
- the ⁇ -acetyl- ⁇ -D-hexosaminidase is referred to simply as acetylglucosinolate, or abbreviated as NAG.
- the N-acetyl- ⁇ -D-hexosinoside of the invention is N-acetyl- ⁇ -D-glucosaside or N-acetyl- ⁇ -D-galactoside.
- the N-acetyl- ⁇ -D-hexosatoside substrate of the present invention has a chromogen, and preferably the chromogen is a bromofluorenyl group (e.g., 5-bromo-4-chloro-3-indolyl), p-nitrobenzene Base, 4-mercapto umbelliferyl or naphthol group.
- chromogen is a bromofluorenyl group (e.g., 5-bromo-4-chloro-3-indolyl acetylglucosamine, p-nitrophenyl-acetylglucosinolate (PNP-NAG), p-nitrophenyl-acetamido Galactoside and the like.
- PNP-NAG p-nitrophenyl-acetamido Galactoside
- Such compounds are commercially available from a number of companies.
- trichomoniasis is an anaerobic organism whose metabolic processes produce large amounts of lower organic amines.
- the inventors have found that in clinical specimens, the pH value of specimens containing trichomoniasis is usually significantly increased to pH 5.0 or higher.
- the present invention relates to a method of detecting the presence of trichomoniasis in a specimen comprising contacting N-acetyl- ⁇ -D-hexosinoside with a specimen.
- N-acetyl- ⁇ -D-hexosaminidase N-acetyl- ⁇ -D-hexosinoside can detect the presence of this enzyme in the specimen.
- the method for detecting trichomoniasis of the present invention comprises: adsorbing N-acetyl having a chromogen
- the detection reagent of ⁇ -D-hexosinoside is contacted with the sample and developed.
- the method of detecting trichomoniasis of the present invention further comprises detecting the pH of the specimen.
- the invention also relates to a method for differentially detecting the presence of trichomoniasis and Candida in a specimen, comprising contacting N-acetyl- ⁇ -D-hexosaminoglycoside with a specimen, and contacting the pH indicator with the specimen to determine the specimen Whether there are trichomoniasis and / or candida.
- N-acetyl- ⁇ -D-aminohexosides can detect the presence of N-acetyl- ⁇ -D-hexosaminidase in the specimen.
- the pH indicator detects the acidity and alkalinity of the specimen.
- the order of the enzyme detecting step and the pH detecting step is not particularly limited, and may be carried out simultaneously.
- the specimen comprises a specimen taken from the vagina, urethra, prostate, or oral cavity.
- a vaginal specimen can be a leucorrhea specimen, such as a vaginal posterior sputum sample.
- the specimen may be diluted with a diluent such as physiological saline. Specimens in contact with the pH indicator can be contacted without dilution.
- N-acetyl- ⁇ -D-hexosaminidase is negatively determined to be free from infection with Trichomonas or Candida, and N-acetyl- ⁇ -D-amino group is used.
- the hexosidase positive is judged to be infected with trichomoniasis or Candida, and if it is found that the pH is not less than 5.0 (preferably not lower than 4.8), it is determined to be infected with trichomoniasis, and if the pH is not higher than 4.5 (preferably not higher than 4.6) ) is judged to be infected with Candida.
- the present invention also relates to a kit comprising N-acetyl- ⁇ -D-hexosinoside as an agent for detecting trichomoniasis.
- the present invention relates to a kit comprising N-acetyl- ⁇ -D-hexosinoside as an agent for detecting trichomoniasis and Candida when N-acetyl- ⁇ -D-hexose When the enzyme is active, it is considered that there are trichomoniasis and/or candida in the specimen.
- the kit of the present invention may further comprise a pH indicator, such as at least one of a variety of pH indicators known in the art, particularly an indicator that distinguishes between pH 4.6 and pH 4.8.
- a pH indicator such as at least one of a variety of pH indicators known in the art, particularly an indicator that distinguishes between pH 4.6 and pH 4.8.
- the pH indicator can be bromophenol green, chlorophenol red, propyl red, and the like.
- kits of the invention may comprise standard colorimetric cards, including standard colorimetric cards for enzymatic reactions (eg, standard colorimetric cards for NAG enzymes, and the The standard colorimetric card for other enzyme reactions) and/or the pH standard color chart.
- the kit of the present invention may be provided with a sensor that senses a color change, such as a sensor that senses a change in color of the NAG and/or a sensor that senses a change in the pH indicator, or may further provide A device that is connected to an electronic device such as a computer.
- the change that occurs after the NAG and/or the pH indicator is in contact with the specimen can be sensed by the sensor, or further the sensory signal can be transmitted to the electronic device, the program in the electronic device recognizes and gives an identification signal, thereby Read NAG detection and / or pH.
- a kit of the present invention may also include a computer readable storage medium, such as an optical disc, for mounting corresponding identification and analysis software.
- kit of the present invention may further comprise a reagent for diluting the specimen, such as physiological saline, and/or a reagent comprising a stop enzyme reaction, for example, a 5-10 mg/ml NaOH-water solution.
- the detection of the invention may be carried out in whole or in part on a solid phase.
- the detection reagents and/or substances of the present invention can be cured on a solid support.
- N-acetyl- ⁇ -D-hexosinoside can be cured at one site of the solid support, and the pH indicator can be cured at another site of the solid support; or N-acetyl- ⁇ -D can be used only.
- the hexosaminoglycoside is cured on a solid support without curing the pH indicator.
- the solid phase carrier may be an adsorption paper, such as filter paper, such as cotton pulp paper, wood pulp paper, sugar cane pulp paper, glass fiber filter paper, etc.; may also be a polyester film, a nylon film, etc.; The ability of plastic.
- filter paper such as cotton pulp paper, wood pulp paper, sugar cane pulp paper, glass fiber filter paper, etc.
- polyester film such as polyester film, a nylon film, etc.
- the kit of the present invention may be a solid phase detecting device such as a test card, a test strip, a test block or the like.
- the solid support of the present invention can be further horizontally covered or embedded in a plastic matrix. Preparation of kit
- the present invention also relates to a method of preparing a kit of the present invention, wherein the preparation of the detection zone for N-acetyl- ⁇ -D-hexosinoside comprises a solution of a chromogen-bearing N-acetyl- ⁇ -D-aminohexose Add to a solid support with adsorption capacity, or will have a suction
- the solid support having the ability is immersed in the solution and then dried, for example, vacuum drying, heat drying, moisture drying, and the like.
- the chromogen-bearing NAG solution is a solution of 1-3 mg/ml chromogen-bearing NAG in phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the chromogenic NAG-containing PBS solution is prepared as follows: The chromogen-bearing NAG is treated with an organic solvent such as decyl alcohol, ethanol, disulfoxide (DMSO) or dimercapto amide (DMF). Dissolve, redissolve in 10-50 mM PBS buffer (pH 5.0-6.0) containing the enzyme inducing agent described above, and mix well.
- organic solvent such as decyl alcohol, ethanol, disulfoxide (DMSO) or dimercapto amide (DMF).
- Dissolve redissolve in 10-50 mM PBS buffer (pH 5.0-6.0) containing the enzyme inducing agent described above, and mix well.
- bromophenol green is used as a pH indicator.
- the bromophenol phenol chloride can be dissolved in absolute ethanol and mixed to form a solution of l-3 mg/ml.
- the prepared bromophenol phenol chloride solution may be added to the solid phase carrier of the present invention, for example, to a paper sheet, or the paper sheet may be immersed in the bromophenol phenol chloride solution, and dried (for example, vacuum drying, heating). After drying, dampness drying, etc., a pH detecting paper is prepared.
- the invention also relates to the use of N-acetyl- ⁇ -D-hexosinoside for the detection of trichomoniasis.
- the present invention also relates to the use of N-acetyl- ⁇ -D-hexosinoside for the preparation of a kit for detecting trichomoniasis. More specifically, the present invention relates to the use of N-acetyl-?-D-hexosinoside for the preparation of a device for detecting trichomoniasis.
- the invention further relates to the use of N-acetyl- ⁇ -D-hexosauroside in combination with a pH indicator for the detection of trichomoniasis or for the differential detection of trichomoniasis and Candida.
- the invention further relates to the use of a combination of N-acetyl- ⁇ -D-hexosinoside and a pH indicator for the preparation of a kit for determining the presence of trichomoniasis and/or Candida. Combined with other vaginitis test methods and kits
- BV Bacterial Vaginosis
- vaginal vaginitis vaginal vaginal vaginitis
- vaginal vaginal vaginitis can detect the production of H 2 0 2 , sialic acid The activity of the enzyme and proline aminopeptidase is judged.
- vaginitis for example, in addition to bacterial vaginosis, Candida vaginitis (also known as mildew) Bacterial vaginitis), unexplained vaginitis other than trichomonas vaginitis, can be judged by detecting leukocyte esterase activity. See, for example, Chinese Patent 200720089307.9.
- the methods and kits of the present invention for detecting trichomoniasis and/or Candida can be further combined with other methods and kits available in the art for detecting other vaginitis (eg, bacterial vaginosis and unexplained vaginitis).
- vaginitis eg, bacterial vaginosis and unexplained vaginitis.
- it is made into a simple and convenient product such as a joint check card, so that end users, not just medical institutions, can quickly, comprehensively and effectively determine the nature of vaginal diseases in a short period of time, in order to timely select effective drug treatment. The disease is convenient.
- the kit of the present invention may additionally comprise other detection reagents, such as reagents known for vaginal disease detection, such as sialidase detection reagent, proline aminopeptidase detection reagent, hydrogen peroxide detection reagent, and Leukocyte esterase detection reagents, and the like.
- detection reagents such as sialidase detection reagent, proline aminopeptidase detection reagent, hydrogen peroxide detection reagent, and Leukocyte esterase detection reagents, and the like.
- At least one of the sialidase detection reagent, the proline aminopeptidase detecting reagent, the hydrogen peroxide detecting reagent, and the leukocyte esterase detecting reagent is cured on a solid phase carrier, for example All are cured on a solid support.
- the curing method is, for example, adding a detection reagent in the form of a solution to a solid phase carrier, or immersing the solid phase carrier in the solution, followed by drying by vacuum, heating, dehumidification or the like.
- the present invention relates to a joint inspection card comprising a card base and a sealing body disposed on the card base, wherein the card base body is respectively provided with an N-acetylhexosaminidase reaction zone and a pH therein In the reaction zone of the reagent, the N-acetylhexosaminidase substrate is solidified with an N-acetylhexosaminidase substrate reagent.
- the sialidase reaction zone and the proline aminopeptidase reaction zone may be further disposed on the card substrate, and the sialidase substrate reagent is solidified in the sialidase reaction zone, and the proline aminopeptidase reaction zone is activated. Cured is a proline aminopeptidase substrate reagent.
- a hydrogen peroxide reaction zone is optionally disposed on the card substrate, and a peroxidase reagent is solidified therein.
- the leukocyte esterase reaction zone may also be optionally disposed on the card substrate, and the leukocyte esterase substrate reagent is solidified therein.
- the sealing body is a sealing cover that is fastened to the side of the notch of the reaction zone on the card base, or a sealing piece adhered to the side of the notch of the reaction zone on the card base. It is of course also possible to use other forms suitable for 7-load liquids (e.g. droplets).
- a method of joint detection according to one aspect of the invention comprising trichomoniasis detection as described above, and/or Candida detection, further comprising at least one assay selected from the group consisting of: sialidase detection, proline aminopeptidase assay, Hydrogen peroxide detection, and leukocyte esterase detection.
- sialidase assay proline aminopeptidase assay, hydrogen peroxide assay, and leukocyte esterase assay are all known in the art.
- the sialidase assay, the proline aminopeptidase assay, the hydrogen peroxide assay, and the leukocyte esterase assay have at least one on a solid support, for example all The solid phase support is carried out.
- vaginal biochemical markers and pH detection of N-acetylhexosaminidase, sialidase, proline aminopeptidase, hydrogen peroxide, leukocyte esterase, etc. are integrated in one joint check card At the same time, these markers are tested to help diagnose the health of the female vagina.
- the vaginal secretions are taken according to the conventional method, and the sample treatment solution can be added for treatment.
- the sample is dropped into each reaction zone of the joint test card, mixed, developed, or added with color development liquid when needed, and the pH value of the sample and the color or/and clarity of the reactants in the tank can be judged. Whether there is vaginal disease, and which kind of vaginosis.
- the N-acetylhexosaminidase reaction is yellow under alkaline conditions, and the proline aminopeptidase reaction is yellow; the sialidase reaction is bright red or purple under the action of the color developing solution; hydrogen peroxide has passed
- the chromogenic substrate oxidizes the chromogenic substrate to produce a red product; the leukocyte esterase reaction appears blue.
- the depth of coloration is proportional to the activity of each enzyme or the concentration of hydrogen peroxide.
- the corresponding reagent or pH adjuster is added to the individual reaction zone.
- Example 1 Preparation of test paper p-Nitrobenzene N-acetyl- ⁇ -D-glucosaminide (purchased from Shanghai Haoyang Biotechnology Co., Ltd., product number M0110) was dissolved in decyl alcohol and dissolved in 25 mM phosphate buffer containing D-galactose. The solution (pH 5.5) was brought to a concentration of 1 mg/ml and mixed well. A 15 ul/piece was added to a 5 mm side paper and dried overnight to prepare a NAG test paper.
- the sodium hydroxide was dissolved in pure water to a concentration of 8 mg/ml, and the mixture was uniformly mixed to obtain a final liquid.
- Bromophenol phenol green was dissolved in absolute ethanol to a concentration of 2 mg/ml, and the mixture was homogenized. It was applied to a paper sheet of 5 mm side length by 15 ul/piece, and dried overnight to prepare a pH-detecting paper sheet.
- the specimen was directly immersed in the pH test paper prepared above to read the pH value, diluted with physiological saline, and dropped onto the NAG test paper prepared above.
- the NAG test paper was heated at 37 °C for 15 minutes, and then the stop solution reagent was added dropwise. NAG detection The yellow color of the paper indicates N-acetyl- ⁇ -D-hexosaminidase activity.
- Trichomonas Two additional samples were separately cultured using Candida chromogenic medium (HiCrome Candida Differential Agar, Himedia Laboratories Pvt. Limited, India) and Trichomonas Medium (OXOID Limited, UK) as described in the manufacturer's instructions. 1. Trichomonas:
- Sensitivity 9 cases of trichomoniasis positive specimens were cultured, and 8 of them were positive by the combined detection method of the present invention, so that the sensitivity of the method for detecting trichomoniasis of the present invention was 88.9% (8/). 9).
- Sensitivity 34 cases of Candida-positive specimens were obtained by culture method, and 26 of them were positive by the combined detection method of the present invention, thereby obtaining the sensitivity of the method of the present invention for detecting Candida was 76.5% (26/ 34).
- NAG test paper and pH test paper were prepared as described in Example 3.
- Conventional reagents for detecting sialidase, proline aminopeptidase, hydrogen peroxide, and leukocyte esterase are separately adsorbed on different sheets of paper, respectively, by methods known in the art. Secure these separate pieces of paper to a plastic card or to a long piece of paper.
- vaginal secretions were taken, diluted, and added to each of the paper sheets prepared in Example 4, and a color developing solution was added to the sialidase paper.
- the entire plastic card or long piece of paper was incubated for 15 minutes at 37 °C.
- the hydrogen peroxide paper is not colored or the light pale color is marked as positive, and the negative label is negative; NAG paper, sialid paper, proline aminopeptidase paper, and leukocyte esterase paper are not colored or The light pastel color is marked as negative, otherwise it is marked as positive.
- the N-acetylhexosaminidase paper is positive, and the paper with the pH reagent is pH indicating pH > 4.8, it is a trichomonas vaginitis infection; if the pH is 4.6, it is indicated as fungal vaginitis.
- the N-acetylhexosaminidase paper is negative, and the lysine aminopeptidase paper is positive, which may indicate BV.
- the sialidase paper is positive, it may be BV.
- the catalase paper is positive, it indicates a vaginal flora imbalance, which may be BV. If the above papers are all negative and only the leukocyte esterase paper is positive, it is indicated as other vaginitis.
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Description
滴虫和念珠菌的检测 技术领域
本发明涉及通过检测 N-乙酰 -β-D-氨基己糖苷酶活性来检测标 本中有无滴虫 (Trichom onas)的方法和试剂盒。 本发明还涉及鉴别性 检测标本中滴虫或念珠菌 (0» ύ¾ώ)的存在的方法和试剂盒。 更进一 步地, 本发明涉及联合检测因滴虫、 念珠菌、 细菌或其它原因引起 的***疾病的方法和试剂盒。 现有技术
已知有三种滴虫可寄生在人体中: ***毛滴虫( vaginalis) , 寄 生在***、 尿道及***, 引起滴虫性***炎等; 人毛滴虫(2: hominis) , 寄生于肠道, 引起肠道滴虫病; 口腔毛滴虫( tewox) , 寄 生于口腔、 齿垢及蛙穴, 引起口腔滴虫病。
目前检查滴虫的方法主要有: 悬滴法, 观察滴虫的运动性, 但 受温度影响较大; 涂片染色法, 将标本处理或不经处理而涂片, 染 色后, 镜检, 此方法受与滴虫大小类似的颗粒的干扰, 并受视野范 围影响; 培养法, 对标本中的滴虫进行针对性的扩增后, 再涂片染 色, 因此耗时长; 免疫学方法, 其用特异性抗体检测滴虫的抗原, 如荧光抗体检查法、 ELISA法、 乳胶凝集法等, 但迄今未发现用于 临床滴虫检测的高品质免疫学产品。 人体中的念珠菌大多属于条件致病菌, 可见于口腔和泌尿生殖 道等处。 目前已知, 85 %以上的条件致病性念珠菌都是白色念珠菌 (C. i//b o»w)和热带念珠菌 (C. tropicalis) , 它们可引起人的泌尿生殖 道疾病。
目前检查念珠菌的方法主要有: 直接涂片镜检法, 女性用消毒 棉拭子取***、 宫颈分泌物或***壁上乳白色薄膜, 男性刮取*** ***、 ***或***处皮损表面鳞屑, 直接涂片镜检, 找卵园形孢
子或找菌丝, 此法有赖于经验, 且受视野、 干扰颗粒特别是取标本 润滑所加的石蜡油影响更大; 真菌培养法, 用专门的培养基例如沙 保氏培养基 (Sabouraud agar, 例 ¾口 Oxoid Ltd., Basingstoke, UK)对标 本中的念珠菌进行针对性扩增, 耗时较长; 乳胶凝集试验, 用多克 隆抗体与念珠菌的甘露聚糖发生反应, 但未见能用于临床的高品质 产品。
滴虫和念珠菌引起的疾病直接从症状上难以鉴别, 治疗用药也 各不相同, 因此需要简单、 快速、 有效的鉴别手段。 发明简述
本发明一方面涉及试剂盒, 尤其例如下列各项描述的试剂盒:
1.1. 试剂盒, 包含 N-乙酰 -β-D-氨基己糖苷作为检测滴虫的试 剂。
1.2. 第 1.1项的试剂盒, 还包括 pH指示剂。
1.3. 第 1.2项的试剂盒, 其中的 N-乙酰 -β-D-氨基己糖苷进一步 任选作为检测念珠菌的试剂。
1.4. 上述任一项的试剂盒, 所述 N-乙酰 -β-D-氨基己糖苷是 N- 乙酰 -β-D-氨基葡萄糖苷或 N-乙酰 -β-D-氨基半乳糖苷。
1.5. 上述任一项的试剂盒, 所述 N-乙酰 -β-D-氨基己糖苷带有 色原, 优选该色原是溴吲哚基、 对硝基苯基、 4-曱基伞形酮或萘酚 基。
1.6. 上述任一项的试剂盒, 还包括至少一种选自下组的试剂: 检测唾液酸苷酶的试剂, 检测脯氨酸氨肽酶的试剂, 检测过氧化氢 的过氧化物酶试剂, 和检测白细胞酯酶的试剂等。
1.7. 上述任一项的试剂盒, 还包括标准比色卡, 例如 NAG标 准比色卡和 /或 pH值标准比色卡。
1.8. 上述任一项的试剂盒, 其中, 选自 N-乙酰 -β-D-氨基己糖 苷, 检测唾液酸苷酶的试剂, 检测脯氨酸氨肽酶的试剂, 过氧化物 酶试剂,检测白细胞酯酶的试剂,和 pH值指示剂的至少一种试剂固 化在固相载体上,优选所述固相载体选自具有吸附能力的纸和塑料,
所述纸例如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸。
1.9. 上述任一项的试剂盒, 为联检卡, 包括卡基体和设置在卡 基体上的密封体。
1.10. 第 1.9项的试剂盒, 所述的密封体为扣合在卡基体上反应 区槽口一侧的密封盖。
1.11. 第 1.9项的试剂盒, 所述的密封体为粘合在卡基体上反应 区槽口一侧的密封片。 本发明另一方面涉及用途, 尤其例如下列各项描述的用途:
2.1. N-乙酰 -β-D-氨基己糖苷用于检测滴虫的用途。
2.2. N-乙酰 -β-D-氨基己糖苷和 pH指示剂联合用于确定滴虫或 念珠菌的存在的用途。
2.3. N-乙酰 -β-D-氨基己糖苷制备检测滴虫的试剂盒的用途。
2.4. N-乙酰 -β-D-氨基己糖苷和 pH指示剂联合用于制备试剂盒 的用途, 所述试剂盒用于确定滴虫或念珠菌的存在。
2.5. 上述任一项的用途, 所述 N-乙酰 -β-D-氨基己糖苷是 N-乙 酰 -β-D-氨基葡萄糖苷或 N-乙酰 -β-D-氨基半乳糖苷。
2.6. 上述任一项的用途, 所述 N-乙酰 -β-D-氨基己糖苷带有色 原, 优选该色原是溴吲哚基、 对硝基苯基、 4-曱基伞形酮或萘酚基。
2.7. 上述任一项的用途, 所述 N-乙酰 -β-D-氨基己糖苷固化在 固相载体上, 优选所述固相载体选自具有吸附能力的纸和塑料, 所 述纸例如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸。 本发明又一方面涉及检测方法,尤其例如下列各项描述的方法:
3.1. 检测标本中滴虫的存在的方法, 包括将 N-乙酰 -β-D-氨基 己糖苷与标本接触。
3.2. 鉴别性检测标本中滴虫和念珠菌的存在的方法, 包括将 N- 乙酰 -β-D-氨基己糖苷与标本接触, 也将 pH指示剂与标本接触。
3.3. 上述任一项的方法, 所述标本是来自***、 尿道、 ***、 或口腔的标本。
3.4. 上述任一项的方法, 所述 N-乙酰 -β-D-氨基己糖苷是 N-乙 酰 -β-D-氨基葡萄糖苷或 N-乙酰 -β-D-氨基半乳糖苷。
3.5. 上述任一项的方法, 所述 N-乙酰 -β-D-氨基己糖苷带有色 原, 优选该色原是溴吲哚基、 对硝基苯基、 4-曱基伞形酮或萘酚基。
3.6. 上述任一项的方法, 所述 N-乙酰 -β-D-氨基己糖苷固化在 固相载体上, 优选所述固相载体选自具有吸附能力纸和塑料, 所述 纸例如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸。
3.7. 上述任一项的方法, 将 N-乙酰 -β-D-氨基己糖苷反应阴性 判为没有滴虫或念珠菌, 或将 N-乙酰 -β-D-氨基己糖苷反应阳性且 H值不低于 4.8判为有滴虫, 或将 N-乙酰 -β-D-氨基己糖苷反应阳 性且 pH值不高于 4.6判为有念珠菌。
3.8. 联合检测方法, 包括进行上述任一项的方法, 并进行至少 一种选自下组的检测: 唾液酸苷酶检测, 脯氨酸氨肽酶检测, 过氧 化氢检测, 和白细胞酯酶检测等
3.9. 第 3.8项的联合检测方法, 其中所述唾液酸苷酶检测, 脯 氨酸氨肽酶检测, 过氧化氢检测, 和白细胞酯酶检测有至少一种在 固相载体上进行。
3.10. 上述任一项的方法, 其中, 选自 N-乙酰 -β-D-氨基己糖苷 酶反应、 唾液酸苷酶反应、 脯氨酸氨肽酶反应、 过氧化氢反应、 白 细胞酯酶反应、 pH指示的至少一种反应, 是通过标本的颜色与各自 的标准比色卡进行比较来判定, 或通过机读仪读取标本的颜色来判 定。 发明内容
酶
在本发明中, N-乙酰 -β-D-氨基己糖苷酶是指, 按照国际生物化 学联合会命名委员会 (Nomenclature Committee of the International Union of Biochemistry)的命名规则, 被分类为 EC.3.2.1.52的酶。 它 主要作用于 N-乙酰 -β-D-氨基葡萄糖苷底物和 N-乙酰 -β-D-氨基半乳 糖苷底物, 水解 N-乙酰 -β-D-氨基己糖苷的末端非还原 N-乙酰 -D-氨
基己糖。 这种酶是诱导酶, 添加该酶的识别基团的类似物能诱导该 酶的分泌。在本发明中,酶诱导剂是指,能诱导本发明的 N-乙酰 -β-D- 氨基己糖苷酶分泌的物质, 可以选自半乳糖、 葡萄糖、 果糖等。
这种酶的别名还有 4艮多, 例如己糖苷酶 (hexosaminidase); β-乙 酰氨基脱氧己糖苷酶 (β-acetylaminodeoxyhexosidase); Ν-乙酰 -β-D- 己糖苷酶 (N-acetyl-p-D-hexosaminidase); N-乙酰 -beta-氨基己糖苷酶 (N-acetyl-beta-hexosaminidase); β-氛基己糖苷酶 (β-hexosaminidase); β-乙酰氨基己糖苷酶 (β-acetylhexosaminidinase); β-D-N-乙酰氨基己 糖苷酶(β-D-N-acetylhexosaminidase); β-N-乙酰氨基葡萄糖苷酶 (β-N-acetylglucosaminidase) ; β-N- 乙 酰 -D-氨基 己 糖 苷 酶 (β-N-acetyl-D-hexosaminidase); 氛基己糖苷酶 A (hexosaminidase A); N-乙酰氨基己糖苷酶 (N-acetylhexosaminidase); β-D-氨基己糖苷酶 (β-D-hexosaminidase) , 它们都包括在本发明范围内。
现已知, 白色念珠菌和热带念珠菌都具有这种酶。 中国专利第 200720089307.9号记载了用 N-乙酰氨基己糖酶法检测霉菌性***炎 (又称念珠菌***炎),该文献全文引入本文作为参考。但迄今未见有 任何关于在临床标本中不经纯化直接检测到带有该酶的滴虫的报 道。
本发明人意外发现, 通过检测 N-乙酰 -β-D-氨基己糖苷酶, 可以 把滴虫及绝大多数念珠菌检测出来。 底物
作为本发明所述 N-乙酰 -β-D-氨基己糖苷酶的底物的化合物也 有别名, 例如 β-乙酰氨基己糖苷; N-乙酰 -β-D-氨基己糖苷; N-乙酰 -beta-氨基己糖苷; β-氨基己糖苷; β-D-N-乙酰氨基己糖苷; β-N-乙 酰氨基葡萄糖苷; β-N-乙酰 -D-氨基己糖苷; Ν-乙酰氨基己糖苷; 或 β-D-氨基己糖苷的化合物等。 用这些名称命名的化合物当被用来 作为如本发明所述的 Ν-乙酰 -β-D-氨基己糖苷酶的底物时,它们都包 括在本发明的范围内。 在实施方案中, 将 Ν-乙酰 -β-D-氨基己糖苷酶 简称为乙酰氨基葡萄糖苷, 或缩写为 NAG。
在实施方案中, 本发明的 N-乙酰 -β-D-氨基己糖苷是 N-乙酰 -β-D-氨基葡萄糖苷或 N-乙酰 -β-D-氨基半乳糖苷。
本发明的 N-乙酰 -β-D-氨基己糖苷底物带有色原,优选该色原是 溴吲哚基 (例如 5-溴 -4-氯 -3-吲哚基)、 对硝基苯基、 4-曱基伞形酮基 或萘酚基。 这样的底物包括, 但不限于, 5-溴 -4-氯 -3-吲哚-乙酰氨基 葡萄糖苷,对硝基苯 -乙酰氨基葡萄糖苷 (PNP-NAG),对硝基苯 -乙酰 氨基半乳糖苷等。 这样的化合物可以在多家公司购买得到。 当色原 是对硝基苯基或萘酚基时, 在反应体系中加入终止液以便及时终止 显色反应。 pH值
本领域已知, 滴虫属于厌氧生物, 其代谢过程产生大量的低级 有机胺。 本发明人发现, 在临床标本中, 含有滴虫的标本 pH值通常 会明显升高, 达到 pH 5.0甚至更高。
本领域还已知, 含有念珠菌的标本 pH值通常在 pH 4.5以下。 本发明人对 1362例临床标本进行统计发现,感染了念珠菌的标本中 pH值等于或低于 4.6的占 94.8 %。
虽然本发明人还发现, 临床上会有念珠菌和滴虫同时感染, 表 现为, 乙酰氨基己糖苷酶阳性、 pH值等于或略低于 4.6。 但这种情 况的发生概率极低, 甚至比本发明人发现的念珠菌标本 pH值大于 4.6的概率还低。 因此, 本发明人考虑, 通过检测 N-乙酰 -β-D-氨基 己糖苷酶,并结合 pH值测定,可以把感染滴虫的标本和感染念珠菌 的标本区别开来。 检测方法
相应地, 本发明涉及检测标本中滴虫的存在的方法, 包括用 N- 乙酰 -β-D-氨基己糖苷与标本接触。 N-乙酰 -β-D-氨基己糖苷作为 N- 乙酰 -β-D-氨基己糖苷酶的底物, 可以检测标本中此酶的存在。 更具 体地, 本发明检测滴虫的方法包括, 将吸附了带有色原的 N-乙酰
-β-D-氨基己糖苷的检测试剂与标本接触并显色。 本发明的检测滴虫 的方法还包括检测标本的 pH值。
本发明还涉及鉴别性检测标本中滴虫和念珠菌的存在的方法, 包括将 N-乙酰 -β-D-氨基己糖苷与标本接触, 并将 pH指示剂与标本 接触, 以此确定标本中是否有滴虫和 /或念珠菌。 N-乙酰 -β-D-氨基己 糖苷可以检测标本中 N-乙酰 -β-D-氨基己糖苷酶的存在。 pH指示剂 可检测标本的酸碱性质。
在本发明的方法中,酶检测步骤和 pH值检测步骤的先后顺序没 有特别限制, 也可以同时进行。
在本发明的方法中, 标本包括取自***、 尿道、 ***、 或口 腔的标本。 例如, ***标本可以是白带标本, 如***后穹窿取样标 本。 标本可以用稀释液稀释, 所述稀释液例如生理盐水。 与 pH指示 剂接触的标本可不经稀释, 直接接触。
在一个具体实施方案中, 通过本发明的方法检测标本后, 将 N- 乙酰 -β-D-氨基己糖苷酶阴性判定为没有感染滴虫或念珠菌, 将 N- 乙酰 -β-D-氨基己糖苷酶阳性判定为感染滴虫或念珠菌, 若另又发现 pH值不低于 5.0(优选不低于 4.8)则判定为感染滴虫, 若 pH值不高 于 4.5(优选不高于 4.6)则判定为感染念珠菌。 试剂盒
本发明还涉及试剂盒,其中包含 N-乙酰 -β-D-氨基己糖苷作为检 测滴虫的一种试剂。 另一方面, 本发明还涉及试剂盒, 其中包含 N- 乙酰 -β-D-氨基己糖苷作为检测滴虫和念珠菌的一种试剂,当捡出 N- 乙酰 -β-D-氨基己糖苷酶活性时, 可认定标本中存在滴虫和 /或念珠 菌。
本发明的试剂盒中还可包含 pH指示剂,例如本领域已知的多种 pH指示剂中的至少一种, 特别是能区分 pH4.6和 pH 4.8的指示剂。 例如, pH指示剂可以是溴曱酚绿, 氯酚红, 丙基红等。
在实施方案中, 本发明的试剂盒可以包含标准比色卡, 包括针 对酶反应的标准比色卡 (如 NAG酶的标准比色卡, 以及下文中提到
的其它酶反应的标准比色卡)和 /或 pH值标准比色卡。
在另外的实施方案中, 本发明的试剂盒可以配有感应颜色变化 的感应器,例如感应 NAG颜色变化的感应器和 /或感应 pH值指示剂 的变化的感应器, 或者还可以提供进一步与计算机等电子设备连线 的设备。 NAG和 /或 pH值指示剂与标本接触后发生的变化可通过所 述感应器感知, 或者进一步可将感知信号传送给所述电子设备, 由 电子设备中的程序识别并给出识别信号, 从而读取 NAG检测值和 / 或 pH值。 因此,本发明的试剂盒中还可以包括用于安装相应的识别 和分析软件的计算机可读存储介质, 如光盘。
另外, 本发明的试剂盒中还可包含稀释标本的试剂, 例如生理 盐水, 和 /或包括终止酶反应的试剂 , 例如 5-10mg/ml的 NaOH-水溶 液。 固相
在实施方案中, 本发明的检测可以全部地或部分地在固相上实 施。 为此, 本发明的检测试剂和 /或物质可被固化在固相载体上。 例 如, 可以将 N-乙酰 -β-D-氨基己糖苷固化在固相载体的一个部位, 并 将 pH指示剂固化在固相载体的另一部位; 也可以仅将 N-乙酰 -β-D- 氨基己糖苷固化在固相载体上, 而并不对 pH指示剂进行固化。
所述固相载体可以是有吸附能力的纸, 例如滤纸, 如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸等; 也可以是聚酯膜、 尼龙膜等; 还可以是有吸附能力的塑料。
相应地, 本发明的试剂盒可以是固相检测用具, 例如检测卡, 检测条, 检测块等等。 本发明的固相载体可进一步水平覆盖或内置 嵌入塑料基体。 试剂盒的制备
本发明还涉及制备本发明试剂盒的方法, 其中, 对于 N-乙酰 -β-D-氨基己糖苷检测区的制备, 包括将带有色原的 N-乙酰 -β-D-氨 基己糖苷的溶液添加到具有吸附能力的固相载体上, 或者将具有吸
附能力的固相载体浸泡在所述溶液中, 然后干燥, 例如真空干燥、 加热干燥、 抽湿干燥等。 优选地, 所述带有色原的 NAG 溶液是 l-3mg/ml带有色原的 NAG在磷酸盐緩冲液 (PBS)中的溶液。 在具体 实施方案中, 所述带有色原的 NAG的 PBS溶液如下制备: 将带有 色原的 NAG用曱醇、乙醇、二曱亚砜 (DMSO)或二曱基曱酰胺 (DMF) 等有机溶剂溶解, 再溶于含有上文所述酶诱导剂的 10-50 mM PBS 緩冲液 (pH 5.0-6.0)中, 混合均匀。
在本发明试剂盒的一个具体实施方案中,釆用溴曱酚绿作为 pH 值指示剂。 可以将溴曱酚氯用无水乙醇溶解, 混合均勾, 制得 l-3mg/ml的溶液。 进一步地, 可以将制得的溴曱酚氯溶液加到本发 明的固相载体上, 例如加到纸片上, 或将纸片浸泡在该溴曱酚氯溶 液中, 干燥 (例如真空干燥、 加热干燥、 抽湿干燥等)后, 制得 pH值 检测纸片。 用途
本发明还涉及 N-乙酰 -β-D-氨基己糖苷用于检测滴虫的用途。本 发明还涉及 N-乙酰 -β-D-氨基己糖苷用于制备检测滴虫的试剂盒的 用途。 更具体地, 本发明涉及 N-乙酰 -β-D-氨基己糖苷用于制备检测 滴虫的用具的用途。
本发明还涉及 N-乙酰 -β-D-氨基己糖苷和 pH指示剂联合用于检 测滴虫或用于鉴别性检测滴虫和念珠菌的用途。 本发明还涉及 N-乙 酰 -β-D-氨基己糖苷和 pH指示剂联合用于制备试剂盒的用途, 所述 试剂盒用于确定滴虫和 /或念珠菌的存在。 与其它***炎检测方法和试剂盒的联合
除了滴虫和念珠菌以外, 在妇女的***中, 还可能因其它原因 造成疾病。 常见的例如细菌性***病 (Bacterial Vaginosis, 简称 BV), 又称非特异性***炎、 加得纳菌性***炎、 ***嗜血杆性***炎, 可通过检测 H202的产生、 唾液酸酶和脯氨酸氨肽酶的活性来判断。 还有例如其它***炎, 即除了细菌性***病, 念珠菌***炎 (又称霉
菌性***炎), 滴虫性***炎等以外的不明原因的***炎, 可通过检 测白细胞酯酶活性来判断。 参见例如中国专利 200720089307.9。
因此, 可以将本发明检测滴虫和 /或念珠菌的方法和试剂盒进一 步地与本领域现有的检测其它***炎 (例如细菌性***病和不明原 因的***炎)的其它方法和试剂盒组合起来, 制成简单方便的联检卡 之类的产品, 以便于最终用户, 而不仅仅是医疗机构, 能够在短时 间内快速、 全面而有效地确定***疾病的性质, 为及时选用有效药 物治疗疾病提供方便。
为此, 本发明的试剂盒可额外包含其它检测试剂, 例如已知用 于***疾病检测的试剂, 像唾液酸苷酶检测试剂, 脯氨酸氨肽酶检 测试剂, 过氧化氢检测试剂, 和白细胞酯酶检测试剂等。
在具体实施方案中, 所述的唾液酸苷酶检测试剂, 脯氨酸氨肽 酶检测试剂, 过氧化氢检测试剂, 和白细胞酯酶检测试剂有至少一 种被固化在固相载体上, 例如全都固化在固相载体上。 所述固化方 式例如, 将溶液形式的检测试剂添加到固相载体上, 或者将固相载 体浸泡在该溶液中, 然后通过真空、 加热、 抽湿等方式进行干燥。
为达到上述目的, 本发明涉及一种联检卡, 包括卡基体和设置 在卡基体上的密封体, 所述的卡基体上分别设置有 N-乙酰氨基己糖 苷酶反应区和其内盛装有 pH试剂的反应区, N-乙酰氨基己糖苷酶 反应区内固化有 N-乙酰氨基己糖苷酶底物试剂。
所述的卡基体上还可选设有唾液酸酶反应区和脯氨酸氨肽酶反 应区, 唾液酸酶反应区内固化有唾液酸酶底物试剂, 脯氨酸氨肽酶 反应区内固化的是脯氨酸氨肽酶底物试剂。
所述的卡基体上还可选设有过氧化氢反应区, 其内固化的是过 氧化物酶试剂。
所述的卡基体上还可选设有白细胞酯酶反应区, 其内固化的是 白细胞酯酶底物试剂。
所述的密封体为扣合在卡基体上反应区槽口一侧的密封盖, 或 为粘合在卡基体上反应区槽口一侧的密封片。 当然也可以釆用其它 适于 7 载液体 (例如液滴)的形式。
本发明一个方面联合检测方法, 包括如上所述的滴虫检测, 和 / 或念珠菌检测, 还包括至少一种选自下组的检测: 唾液酸苷酶检测, 脯氨酸氨肽酶检测, 过氧化氢检测, 和白细胞酯酶检测等。 所述的 唾液酸苷酶检测, 脯氨酸氨肽酶检测, 过氧化氢检测, 和白细胞酯 酶检测都是本领域已知的。 相应地, 在具体实施方案中, 所述的唾 液酸苷酶检测, 脯氨酸氨肽酶检测, 过氧化氢检测, 和白细胞酯酶 检测有至少一种在固相载体上进行, 例如全都在固相载体上进行。
在一个具体实施方案中, 将 N-乙酰氨基己糖苷酶、 唾液酸酶、 脯氨酸氨肽酶、 过氧化氢、 白细胞酯酶等五个***生化标志物和 pH 值检测整合在一个联检卡上, 同时对这些标志物进行检测, 辅助诊 断女性***的健康状况。
检测前, 先按照常规方法釆取***分泌物, 可加入样品处理液 进行处理。 将样品滴入联检卡的各个反应区内, 混匀, 显色, 或需 要时补加显色液显色 , 结合样品的 pH值以及槽内反应物的颜色或 / 和澄清度变化就能够判断有无***病, 以及是哪一种***病。 例如, N-乙酰氨基己糖苷酶反应在碱性条件下显黄色, 脯氨酸氨肽酶反应 呈现黄色; 唾液酸酶反应在显色液的作用下呈鲜红色或紫色; 过氧 化氢在过氧化物酶的催化下可以氧化生色底物, 产生红色的产物; 白细胞酯酶反应呈现蓝色。 呈色深度与各酶活性或过氧化氢浓度成 正比。在实际检测时,个别反应区内补加相应的显色剂或 pH值调节 剂。
从上述可以看出, 在联检卡上仅设置 N-乙酰氨基己糖苷酶反应 区和 pH试剂的反应区,并在槽内固化相应的试剂就能够鉴别滴虫或 念珠菌感染。 同时在联检卡上增设唾液酸酶、 脯氨酸氨肽酶、 过氧 化氢和 /或白细胞酯酶的反应区, 便于同时检测这些***生化标志 物, 以进一步区分细菌性***病、 霉菌性***炎、 滴虫性***炎及 其他***炎。 实施例
实施例 1 制备检测纸片
对硝基苯 N-乙酰 -β-D-氨基葡萄糖苷 (购自上海楷洋生物技术有 限公司, 产品编号 M0110)用曱醇溶解后, 再溶于含有 D-半乳糖的 25mM磷酸盐緩冲液 (pH5.5),使浓度达到 lmg/ml,混合均匀。按 15ul/ 片加到 5mm边长的纸片上, 干燥过夜, 制得 NAG检测纸片。
氢氧化钠用纯水溶解, 使浓度达到 8mg/ml, 混合均匀, 制得终 止液。
溴曱酚绿用无水乙醇溶解, 使浓度达到 2mg/ml, 混合均勾。 按 15ul/片加到 5mm边长的纸片上, 干燥过夜, 制得 pH值检测纸片。 实施例 2 pH值检测
截止 2009年,从郑州大学第三附属医院妇科门诊获得妇科白带 标本 10000多例 ,用上述制备的 pH值检测纸片来检测标本的 pH值, 结果发现, 感染滴虫的标本 pH值都不低于 4.8, 而感染念珠菌的标 本的 pH值都不高于 4.6。 实施例 3 酶检测联合 pH值检测
郑州大学第三附属医院妇科门诊 212例患者, 每位都用无菌拭 子于***后穹窿取标本。
1. 本发明的检测
先将标本直接在上述制得的 pH检测纸片蘸一蘸以读取 pH值, 再用生理盐水稀释后滴加在上述制备的 NAG检测纸片上。 将 NAG 检测纸片放在 37 °C加热 15分钟 , 然后滴加终止液试剂。 NAG检测 纸片显黄色表明具有 N-乙酰 -β-D-氨基己糖苷酶活性。
2. 常规检测
另取两份标本分别釆用念珠菌显色培养基 (HiCrome Candida Differential Agar, Himedia Laboratories Pvt. Limited, 印度)和滴虫培 养基 (Trichomonas Medium, OXOID Limited,英国)按照厂家说明书的 描述进行培养。
1. 滴虫:
(1) 灵敏度: 培养法捡出滴虫阳性标本 9例,其中有 8例同时用 本发明的联合检测方法测出为阳性, 从而得出本发明方法检测滴虫 的灵敏度为 88.9% (8/9)。
(2) 特异性: 培养法捡出滴虫阴性标本 203例,其中有 198例同 时用本发明的联合检测方法测出为阴性, 从而得出本发明方法检测 滴虫的特异性为 97.7% (198/203)。
2. 念珠菌:
(1) 灵敏度: 培养法捡出念珠菌阳性标本 34例,其中有 26例同 时用本发明的联合检测方法测出为阳性, 从而得出本发明方法检测 念珠菌的灵敏度为 76.5% (26/34)。
(2) 特异性: 培养法捡出念珠菌阴性标本 178 例, 其中有 149 例同时用本发明的联合检测方法测出为阴性, 从而得出本发明方法 检测念珠菌的特异性为 85.6% (149/178)。 实施例 4 制备联检试剂盒
按实施例 3所述方法, 制得 NAG检测纸片和 pH检测纸片。 按 本领域已知方法, 将检测唾液酸苷酶、 脯氨酸氨肽酶、 过氧化氢、 和白细胞酯酶的常规试剂分别吸附在不同的纸片上。 将这些相互独 立的纸片固定在塑料卡上或粘在长纸片上。 实施例 5 妇科联检
方案 1 :
取***分泌物, 稀释后, 滴加至实施例 4制得的各个纸片上, 并在唾液酸苷酶纸片上滴加显色液。
1分钟后, 对照比色卡记录 pH值。
再将整个塑料卡或长纸片至于 37 °C温育 15分钟。
在 NAG纸片上滴加终止液, 30秒后, 判读除 pH值以外的其它 反应结果。
方案 2:
与方案 1的区别仅在于: 用机读仪读取 pH值。 为此, pH检测 区在温育之前不加样, 待温育后再加入经稀释或未经稀释的标本。 结果记录:
过氧化氢纸片不显色或显浅淡色标记为阳性,反之标记为阴性; NAG纸片、 唾液酸酶纸片、 脯氨酸氨肽酶纸片、 以及白细胞酯酶纸 片不显色或显浅淡色标记为阴性, 反之标记为阳性。 结果解释:
例如, N-乙酰氨基己糖苷酶纸片显阳性, 同时固化有 pH试剂 的纸片指示 pH > 4.8, 则为滴虫性***炎感染; 若同时 pH 4.6, 则 指示为霉菌性***炎。 N-乙酰氨基己糖苷酶纸片显阴性, 同时脯氨 酸氨肽酶纸片显阳性, 则可能指示 BV。 唾液酸酶纸片显阳性, 则可 能是 BV。 若过氧化氢酶纸片显阳性则指示***菌群失调, 可能是 BV。 若果上述纸片均显阴性, 而仅有白细胞酯酶纸片显阳性, 则指 示为其他***炎。
Claims
1. 试剂盒, 包含 N-乙酰 -β-D-氨基己糖苷作为检测滴虫的试剂。
2.权利要求 1的试剂盒,还包括 pH指示剂,其中的 N-乙酰 -β-D- 氨基己糖苷进一步任选作为检测念珠菌的试剂。
3. 权利要求 1或 2的试剂盒, 所述 N-乙酰 -β-D-氨基己糖苷带 有色原, 优选该色原是溴吲哚基、 对硝基苯基、 4-曱基伞形酮或萘 酚基。
4.权利要求 1或 2的试剂盒,还包括至少一种选自下组的试剂: 检测唾液酸苷酶的试剂, 检测脯氨酸氨肽酶的试剂, 检测过氧化氢 的过氧化物酶试剂, 和检测白细胞酯酶的试剂等。
5. 权利要求 1或 2的试剂盒, 其中, 选自 N-乙酰 -β-D-氨基己 糖苷, 检测唾液酸苷酶的试剂, 检测脯氨酸氨肽酶的试剂, 过氧化 物酶试剂,检测白细胞酯酶的试剂,和 pH值指示剂的至少一种试剂 固化在固相载体上, 优选所述固相载体选自具有吸附能力的纸和塑 料, 所述纸例如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸。
6. N-乙酰 -β-D-氨基己糖苷制备检测滴虫的试剂盒的用途。
7. N-乙酰 -β-D-氨基己糖苷和 pH指示剂联合用于制备试剂盒的 用途, 所述试剂盒用于确定滴虫或念珠菌的存在。
8. 权利要求 6或 7的用途, 所述 N-乙酰 -β-D-氨基己糖苷是 N- 乙酰 -β-D-氨基葡萄糖苷或 N-乙酰 -β-D-氨基半乳糖苷。
9. 权利要求 6或 7的用途, 所述 N-乙酰 -β-D-氨基己糖苷带有 色原, 优选该色原是溴吲哚基、 对硝基苯基、 4-曱基伞形酮或萘酚 基。
10. 权利要求 6或 7的用途, 所述 N-乙酰 -β-D-氨基己糖苷固化 在固相载体上, 优选所述固相载体选自具有吸附能力的纸和塑料, 所述纸例如棉浆纸、 木浆纸、 甘蔗浆纸和玻璃纤维滤纸。
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CN102253206A (zh) * | 2011-06-23 | 2011-11-23 | 泰普生物科学(中国)有限公司 | 一种***炎联合检测试剂盒 |
CN102288746A (zh) * | 2011-07-12 | 2011-12-21 | 英科新创(厦门)科技有限公司 | 一种血液筛查联合检测卡 |
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CN106226301A (zh) * | 2016-08-22 | 2016-12-14 | 长沙励思生物技术有限公司 | ***炎多联检测卡 |
CA3089526A1 (en) * | 2018-02-07 | 2019-08-15 | Mycometer A/S | Improved method for measurement of microbial biomass |
CN113450354B (zh) * | 2021-08-30 | 2021-11-16 | 山东仕达思生物产业有限公司 | 基于卷积神经网络和摇摆活动特征的滴虫检测方法 |
CN117106938B (zh) * | 2023-08-31 | 2024-05-28 | 苏州创澜生物科技有限公司 | 一种用于检测***炎病原体的试剂、试剂盒及应用 |
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- 2010-01-27 ES ES10750325T patent/ES2436372T3/es active Active
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Also Published As
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US20150167049A1 (en) | 2015-06-18 |
ES2436372T3 (es) | 2013-12-30 |
EP2407549B1 (en) | 2013-10-09 |
CN201400688Y (zh) | 2010-02-10 |
EP2407549A1 (en) | 2012-01-18 |
EP2407549A4 (en) | 2012-07-25 |
US20120034637A1 (en) | 2012-02-09 |
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