CN112946297A - Diagnostic marker for decomposing mycoplasma urealytium infection and preparation method and application of corresponding detection kit thereof - Google Patents

Abstract

The invention discloses a diagnostic marker for ureaplasma urealyticum infection and a preparation method and application of a corresponding detection kit thereof, wherein the diagnostic marker is polypeptide containing UUR 10-0579 protein antigenic determinant sites, and the amino acid sequence of the polypeptide is shown as sequence 1. The invention detects pathogen-related specific antibodies by quantitative ELISA, and has the advantages of rapidness, convenience, high flux and high result repeatability; secondly, the UUR10_0579 protein is an important antigen for the early infection of the mycoplasma urealytium and is involved in the immune response of organisms, so that the UUR10_0579 protein can be used as a specific diagnostic marker for the infection of the mycoplasma urealytium. Compared with the current clinical detection method for ureaplasma urealyticum, the detection method for ureaplasma urealyticum infection provided by the invention has the advantages of early stage, rapidness, sensitivity, specificity, accuracy and the like, and makes up for the defects of the current clinical detection method. The detection method has objective result judgment and simple and convenient operation, is suitable for large-scale screening test, and can be used for final confirmation diagnosis of ureaplasma urealyticum infection.

Description

Diagnostic marker for decomposing mycoplasma urealytium infection and preparation method and application of corresponding detection kit thereof

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a diagnostic marker for decomposing mycoplasma urealytium infection and a preparation method and application of a corresponding detection kit thereof.

Background

Ureaplasma Urealyticum (UU), also known as ureaplasma urealyticum, is one of six ureaplasma genera, and has close relationship with human urogenital tract infection. The bacterial cells are tiny, only one fifth of that of Escherichia coli, and are the smallest and simplest prokaryotes with self-reproduction capability known at present. UU is mainly distributed in the urogenital tract of a human body and is the most common pathogenic bacterium of non-gonococcal urethritis except chlamydia trachomatis. It can invade female genital tract mucous membrane cell to cause vaginitis, cervicitis, continue ascending infection, adsorb on oviduct mucous membrane to cause cilia to drop off, epithelial damage, salpingitis, endometritis, etc., and can also cause intrauterine infection through mother-infant vertical transmission to cause abortion, dead fetus, premature birth, neonatal respiratory infection, etc. Prostatitis, epididymitis, urethritis, etc. can be caused after male infection.

In recent years, non-gonococcal urethritis caused by ureaplasma urealyticum infection has become the most common sexually transmitted disease, and the incidence rate is on the rise, so that the detection of ureaplasma urealyticum is of great significance in the diagnosis of urinary tract system diseases.

The current methods for detecting ureaplasma urealyticum in clinic mainly comprise: culture methods, PCR methods, immunological methods, and the like. The culture method is a traditional 'gold standard' method, has high specificity, but has the defects of low sensitivity, long culture time, easy pollution and the like, thereby limiting the clinical application of the method. The PCR method has the characteristics of high sensitivity, high specificity and the like, has superiority in clinical diagnosis, but needs special instruments and special personnel for operation, and is difficult to popularize in primary hospitals. The immunological method has the advantages of rapidness, simplicity, convenience, easy standardization and the like, and has good application prospect in clinical rapid detection. However, the major immunological methods (gold-labeled method and fluorescent antibody method) for detecting ureaplasma urealyticum in clinical practice have certain limitations. At present, the detection of mycoplasma antibody by using an indirect method in China is a qualitative reagent, but because mycoplasma exists widely in people, normal people also have low titer antibody level, diagnosis cannot be carried out only by means of antibody qualitative detection; in addition, the monoclonal antibody immunofluorescence method has more influence factors on the urea-dissolving mycoplasma detection, and false negative results can be caused by the fact that cell components are not taken during smear and are lost during fixation. Thus, a sensitive, rapid, and accurate quantitative detection method is needed.

Enzyme-linked immunosorbent assay (ELISA) is a plurality of pathogens including viruses, bacteria, fungi, mycoplasma and the like, is the most common detection method, has the characteristics of simple and convenient operation, high detection accuracy and the like, and is widely applied to clinic and disease control. The traditional qualitative or semi-quantitative ELISA is low in price and is the first choice detection method for primary hospitals and disease control centers at present. With the development of clinical detection technology, the quantitative ELISA has higher accuracy, can reflect the organism immunity level of patients better, and is gradually applied clinically.

The UUR10_0579 protein is a novel glycosyltransferase, has the molecular weight of 37kDa, can catalyze and synthesize glycolipids, is an important antigen for decomposing early infection of mycoplasma urealytium, and participates in immune response of organisms. Therefore, the mycoplasma urealyticum infection can be diagnosed early by quantitatively detecting the specific antibody of the UUR10_0579 protein.

Disclosure of Invention

The invention aims to provide a diagnostic marker for ureaplasma urealyticum infection and a preparation method and application of a corresponding detection kit thereof, and establishes a novel ureaplasma urealyticum detection technology which can diagnose ureaplasma urealyticum infection objectively, sensitively and stably in an early stage.

The diagnostic marker for mycoplasma urealyticum infection is polypeptide containing UUR 10-0579 protein antigenic determinant site, and the amino acid sequence is shown as sequence 1.

The method for preparing the detection kit from the diagnostic marker for ureaplasma urealyticum infection comprises the following steps:

s1, analyzing the amino acid sequence of UUR10_0579 protein by adopting experimental bioinformatics software, predicting and determining an antigenic determinant site on the UUR10_0579 protein, and artificially combining polypeptides containing the antigenic determinant site, wherein the amino acid sequence of the polypeptides is shown as a sequence 1;

s2, taking the polypeptide artificially synthesized in the step S1 as an antigen, diluting the polypeptide with a coating buffer solution to obtain a diluted polypeptide solution, adding the diluted polypeptide solution into a 96-hole enzyme label plate at 100 mu L/hole, washing the plate for 3 times at 4 ℃ overnight, then adding 200 mu L of phosphate buffer solution containing 1% bovine serum albumin into the diluted polypeptide solution for sealing, incubating the plate for 1h at 37 ℃, washing the plate for 3 times, drying the plate, packaging the plate with a sealing bag to obtain the enzyme label plate coated with the UUR10_0579 protein antigen in advance, and storing the plate for later use;

the components of the S3 ELISA detection kit are as follows: the ELISA plate coated with UUR 10-0579 protein antigen in step S2 in advance; 3mL x 1 bottles of calf serum; a 3.2mL x 1 bottle of the enzyme-labeled antibody is prepared; fourth, 1mL x 1 bottles of positive control serum; fifthly, obtaining 1mL x 1 bottles of negative control serum; sixthly, concentrating a washing solution into a bottle of 30mL x 1; aquacide A4mL x 1 bottles; and developer B4 mL x 1 bottle; a 3.5mL x 1 bottle of a self-supporting agent; the sealing plates are 3; serum sample diluting buffer solution 1 bottle.

In step S2, the coating buffer is 0.05 mol/L carbonate buffer, pH 9.6, and the preparation process comprises mixing 1.59 g sodium carbonate (NaCO)₃) And 2.93 grams of sodium bicarbonate (NaHCO)₃) Dissolving in 1000 ml of distilled water, and uniformly mixing; the coating buffer diluted the polypeptide to 10. mu.g/mL.

In step S2, the phosphate buffer solution is PBS (0.01 mol/L), pH is 7.4, and the preparation process comprises mixing 8.0 g sodium chloride (NaCl), 0.2g potassium dihydrogen phosphate (KH)₂PO₄) 2.9 g disodium hydrogen phosphate (Na)₂HPO_4`12H₂O) and 0.2g of potassium chloride (KCl) are added into 1000 ml of distilled water, and the mixture is dissolved and mixed evenly; sealing liquid: adding 1 g bovine serum albumin into 100 ml PBS, dissolving and mixingAnd (4) homogenizing.

In the step S3, the enzyme-labeled antibody is an enzyme-labeled secondary antibody, preferably a commercial HRP (horse radish peroxidase) -labeled horse anti-mouse IgG; concentrating the washing solution to p H value 7.4 and final concentration of 0.1mol/L PBS solution, adding Tween-20 to make the concentration of Tween-20 0.5% (v/v), and diluting with water 10 times before use; subpackaging according to 30 mL/bottle; the developer A is prepared by adding 13.6g of sodium acetate, 1.6g of citric acid, 0.3mL of 30% hydrogen peroxide and distilled water to 500mL, and subpackaging according to 4 mL/bottle; the color developing agent B is prepared by adding 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid, 50mL of glycerol and 0.15g of TMB distilled water to 500mL, and subpackaging according to 4 mL/bottle; the terminating agent is preferably 2mol/L H₂SO₄(ii) a The serum sample dilution buffer is preferably phosphate buffer with pH 7.2-7.5.

The method for detecting the specific antibody in the UUR10_0579 protein serum by using the detection kit comprises the following steps:

(1) sample adding: taking serum to be detected, adding a serum sample dilution buffer solution for dilution, and uniformly mixing;

(2) and (3) incubation: adding calf serum and one drop of each diluted serum sample to be detected in the step (1) into each hole, setting two holes of negative/positive control, adding two drops of negative control or positive control into each hole, and sealing the plates for 1h at 37 ℃;

(3) washing: the microporous plate is thrown to remove liquid in the pores, washing liquid is injected, the microporous plate is placed for 10s, then is dried by spinning, and the process is repeated for 4 times;

(4) adding an enzyme-labeled antibody: adding 1 drop of enzyme-labeled antibody into each well, and sealing the plate for 1h at 37 ℃;

(5) washing the plate as before;

(6) color development: adding one drop of each of the color developing agent A and the color developing agent B, and standing for 20min at 37 ℃;

(7) and (3) terminating the reaction: adding 1 drop of terminator into each hole;

(9) and observing and recording results: the OD optical density value at 490nm when the excitation light was read was used to calculate the antibody content.

The principle of the invention is as follows: the invention provides a quantitative ELISA antibody detection kit which can quantitatively detect specific antibodies of UUR10_0579 protein in serum of a patient by using a quantitative ELISA principle. Briefly, enzyme-linked immunosorbent assay is an immunoreaction-based assay method in which an antibody is immobilized on a carrier, and an antigen and a catalytic enzyme-labeled antibody are added to perform an immunological binding reaction. When the enzymatic substrate is added, the substrate is enzymatically reacted to form a chromogenic product. The concentration of the chromogenic product is directly or linearly related to the amount of the substance to be detected, so that quantitative calculation or qualitative analysis can be directly performed.

The invention uses UUR10_0579 protein of ureaplasma urealyticum as antigen to coat an enzyme label plate and positive control serum to prepare antigen, prepares a quantitative ELISA antibody detection kit for detecting the specific antibody of UUR10_0579 protein in serum of a patient and carrying out quantitative analysis, and judges whether the patient is infected with ureaplasma urealyticum according to the detection result. The principle diagram is shown in figure 1, the antigen is connected to the solid phase carrier, the antibody to be detected in the sample is combined with the solid phase antigen-detected antibody compound, the enzyme-labeled secondary antibody (the antibody aiming at the detected antibody) is combined with the antibody in the solid phase immune compound to form the solid phase antigen-detected antibody-enzyme-labeled secondary antibody compound, and the color development degree after adding the substrate can be quantitatively measured by visual inspection or a spectrophotometer to determine the content of the antibody to be detected.

The invention has the beneficial effects that: the invention detects pathogen-related specific antibodies by quantitative ELISA, and has the advantages of rapidness, convenience, high flux and high result repeatability; secondly, the UUR10_0579 protein is an important antigen for the early infection of the mycoplasma urealytium and is involved in the immune response of organisms, so that the UUR10_0579 protein can be used as a specific diagnostic marker for the infection of the mycoplasma urealytium. Compared with the current clinical detection method for ureaplasma urealyticum, the detection method for ureaplasma urealyticum infection provided by the invention has the advantages of early stage, rapidness, sensitivity, specificity, accuracy and the like, and makes up for the defects of the current clinical detection method. The detection method has objective result judgment and simple and convenient operation, is suitable for large-scale screening test, and can be used for final confirmation diagnosis of ureaplasma urealyticum infection.

Drawings

FIG. 1 is a schematic diagram of the preparation of the present invention;

FIG. 2 is a flow chart for preparing the kit of the present invention.

Detailed Description

Example 1

The flow chart of the preparation of this example is shown in fig. 2, which includes the following steps:

preparation of UUR10_0579 protein of ureaplasma urealyticum

According to the characteristics of strong antigenicity, good surface property and small molecular weight of the UUR-resolving mycoplasma UUR10_0579 protein, experimental bioinformatics software analyzes the amino acid sequence of the UUR10_0579 protein, predicts and determines an antigenic determinant site on the UUR10_0579 protein, and artificially combines polypeptides (shown as a sequence 1) containing the antigenic determinant site, so that the purity of the purified UUR-resolving mycoplasma UUR is more than 95%.

Sequence 1 the amino acid sequence is as follows:

MLLTIAFYLSKTTHYLKKNFNYFLDLLNQNKKHIELIIIDDASDYNLFKTLKPLIENTNSKIKYFYLNETQGNAYAYNLATKYAHGKYIWYLGGHTELNLDASSLLFSVLEKDYDVISFNLNDNVNQNPSLVFDSLNKEVLVGLWESISNKIIALDFIKKHQLAFYNDKWYPALFIYDLFTKFSSWRNVNVNFISNNSGEVGYNVYDLLQQINELYAKFSNDGLLEIYKDELCYWITGICIHSFLKKIYELYTININSKKQIKERTMIISHALSNAKKYLETYFANFENNPYVRKYKTNILKYYLKSKQGLN

secondly, preparing an ELISA plate pre-coated with UUR 10-0579 protein antigen

The artificially synthesized polypeptide is used as antigen, diluted to 10 mu g/mL by using coating buffer solution respectively, then coated in a 96-well enzyme label plate by 100 mu L/well, and the plate is washed 3 times at 4 ℃ overnight. Adding 200 microliters of phosphate buffer solution containing 1% bovine serum albumin into each hole for sealing, incubating at 37 ℃ for 1 hour, washing the plate for 3 times, drying in the air, packaging the ELISA plate by using a sealing bag, and storing at 4 ℃ for later use.

Preparation of other reagents used for enzyme-labeled plate preparation:

(1) coating buffer (0.05 mol/l carbonate buffer, pH 9.6: 1000 ml distilled water added with 1.59 g sodium carbonate (NaCO)₃) And 2.93 grams of sodium bicarbonate (NaHCO)₃) And dissolving and mixing uniformly.

(2) Phosphate buffer (0.01 mol/L PBS, pH 7.4) 1000 ml distilled water was added with 8.0 g sodium chloride (NaCl), 0.2g potassium dihydrogen phosphate (KH)₂PO₄) 2.9 g disodium hydrogen phosphate (Na)₂HPO_4`12H₂o) and 0.2gPotassium chloride (KCl), and mixing.

(3) Sealing liquid: 100 ml PBS was added with 1 g bovine serum albumin, dissolved and mixed.

Thirdly, determining the components of the quantitative ELISA detection kit for ureaplasma urealyticum

The components of the ELISA detection kit are as follows: (1) 1 microwell reaction plate coated with synthetic antigen polypeptide; (2) calf serum 3mL x 1 bottle; (3) 3.2mL x 1 bottle of an ELISA plate antibody; (4) positive control serum 1mL x 1 bottle; (5) negative control serum 1mL x 1 bottle; (6) concentrating 25 times of washing solution in a 30mL x 1 bottle; (7) 4mL x 1 bottle of color developing agent A; (8) 4mL x 1 bottle of color developing agent B; (9) terminator 3.5mL x 1 bottle; (10) 3 sealing sheets; (11) serum sample dilution buffer 1 bottle.

The enzyme-labeled antibody is an enzyme-labeled secondary antibody, and is specifically a commercial horse anti-mouse IgG labeled by HRP (horse radish peroxidase); concentrating the washing solution to p H value 7.4 and final concentration of 0.1mol/L PBS solution, adding Tween-20 to make the concentration of Tween-20 0.5% (v/v), and diluting with water 10 times before use; subpackaging according to 30 mL/bottle; the developer A is prepared by adding 13.6g of sodium acetate, 1.6g of citric acid, 0.3mL of 30% hydrogen peroxide and distilled water to 500mL, and subpackaging according to 4 mL/bottle; the color developing agent B is prepared by adding 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid, 50mL of glycerol and 0.15g of TMB distilled water to 500mL, and subpackaging according to 4 mL/bottle; the terminating agent is preferably 2mol/L H₂SO₄(ii) a The serum sample dilution buffer is preferably phosphate buffer with pH 7.2-7.5.

The negative control serum contained in the kit is a normal serum sample, and the positive control serum is a serum sample of a patient who is definitely infected with the virus and is verified by experiments.

Example 2

The specific operation process of detecting specific antibodies in serum of UUR10_0579 protein by the kit in example 1 is as follows:

the implementation operation comprises the following specific steps of (1) sample adding: taking serum to be detected, adding a serum sample dilution buffer solution, and uniformly mixing; (2) and (3) incubation: adding one drop of calf serum and one drop of serum specimen to be detected into each hole, setting two holes of negative/positive control, adding two drops of negative control or positive control into each hole, and sealing the plates for 1h at 37 ℃; (3) washing: the microporous plate is thrown to remove liquid in the pores, washing liquid is injected, the microporous plate is placed for 10 seconds, the microporous plate is dried, and the steps are repeated for 4 times; (4) adding an enzyme-labeled antibody: adding 1 drop of enzyme-labeled antibody into each well, and sealing the plate for 1h at 37 ℃; (5) washing the plate as before; (6) color development: adding one drop of each of the color developing agent A and the color developing agent B, and standing for 20min at 37 ℃; (7) and (3) terminating the reaction: adding 1 drop of terminator into each hole; (9) and observing and recording results: the OD optical density value at 490nm when the excitation light was read was used to calculate the antibody content.

In this example, 24 sera were tested, and the OD values and the antibody contents calculated therefrom are shown in table 1; of these, 13 were infected with mycoplasma urealyticum. Antibody content of 1.5129 XOD₄₉₀+3.4803(R²0.992), when the antibody content is more than or equal to 5.00ng/mL, the specimen is determined to be positive, and when the antibody content is less than 5.00ng/mL, the specimen is determined to be negative.

Table 1 OD values and antibody contents of 24 sera examined

The serum is tested by the current PCR method, wherein 13 infectious known ureaplasma urealyticum are contained, and the infectious data measured in the embodiment 2 correspond to one, so that the kit disclosed by the invention has good accuracy.

Sequence listing

<110> Xiangya three hospitals of Zhongnan university

<120> diagnostic marker for ureaplasma urealyticum infection and preparation method and application of corresponding detection kit thereof

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Claims (6)

1. A diagnostic marker for mycoplasma urealytium infection is characterized in that the diagnostic marker is a polypeptide containing UUR10_0579 protein antigenic determinant sites, and the amino acid sequence of the polypeptide is shown as sequence 1.

2. The method for preparing a detection kit from the diagnostic marker for mycoplasma urealytium infection according to claim 1, comprising the steps of:

s1, analyzing the amino acid sequence of UUR10_0579 protein by adopting experimental bioinformatics software, predicting and determining an antigenic determinant site on the UUR10_0579 protein, and artificially combining polypeptides containing the antigenic determinant site, wherein the amino acid sequence of the polypeptides is shown as a sequence 1;

s2, taking the polypeptide artificially synthesized in the step S1 as an antigen, diluting the polypeptide with a coating buffer solution to obtain a diluted polypeptide solution, adding the diluted polypeptide solution into a 96-hole enzyme label plate at 100 mu L/hole, washing the plate for 3 times at 4 ℃ overnight, then adding 200 mu L of phosphate buffer solution containing 1% bovine serum albumin into the diluted polypeptide solution for sealing, incubating the plate for 1h at 37 ℃, washing the plate for 3 times, drying the plate, packaging the plate with a sealing bag to obtain the enzyme label plate coated with the UUR10_0579 protein antigen in advance, and storing the plate for later use;

the components of the S3 ELISA detection kit are as follows: the ELISA plate coated with UUR 10-0579 protein antigen in step S2 in advance; 3mL x 1 bottles of calf serum; a 3.2mL x 1 bottle of the enzyme-labeled antibody is prepared; fourth, 1mL x 1 bottles of positive control serum; fifthly, obtaining 1mL x 1 bottles of negative control serum; sixthly, concentrating a washing solution into a bottle of 30mL x 1; aquacide A4mL x 1 bottles; and developer B4 mL x 1 bottle; a 3.5mL x 1 bottle of a self-supporting agent; the sealing plates are 3; serum sample diluting buffer solution 1 bottle.

3. The method for preparing a detection kit according to the diagnostic marker for mycoplasma urealytium infection of claim 2, wherein in step S2, the coating buffer is 0.05 mol/l carbonate buffer, and the pH is 9.6, and the specific preparation process comprises dissolving 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate in 1000 ml of distilled water, and mixing the solution; the coating buffer diluted the polypeptide to 10. mu.g/mL.

4. The method for preparing a test kit according to claim 2, wherein in step S2, the phosphate buffer solution is 0.01 mol/L PBS, pH 7.4, and the method is specifically performed by adding 8.0 g of sodium chloride, 0.2g of potassium dihydrogen phosphate, 2.9 g of disodium hydrogen phosphate and 0.2g of potassium chloride into 1000 ml of distilled water, and dissolving and mixing the components uniformly; sealing liquid: 100 ml PBS was added with 1 g bovine serum albumin, dissolved and mixed.

5. The method for preparing a detection kit from a diagnostic marker for ureaplasma urealyticum infection according to claim 2, wherein in the step S3, the enzyme-labeled antibody is an enzyme-labeled secondary antibody, specifically, a commercial HRP (horse radish peroxidase) -labeled horse anti-mouse IgG; concentrating the washing solution to p H value 7.4 and final concentration of 0.1mol/L PBS solution, adding Tween-20 to make the concentration of Tween-20 0.5% (v/v), and diluting with water 10 times before use; subpackaging according to 30 mL/bottle; the developer A is prepared by adding 13.6g of sodium acetate, 1.6g of citric acid, 0.3mL of 30% hydrogen peroxide and distilled water to 500mL, and subpackaging according to 4 mL/bottle; the color developing agent B is prepared by adding 0.2g of disodium ethylene diamine tetraacetate, 0.95g of citric acid, 50mL of glycerol and 0.15g of TMB distilled water to 500mL, and subpackaging according to 4 mL/bottle; the terminating agent is 2mol/L H₂SO₄(ii) a The serum sample dilution buffer is phosphate buffer with pH 7.2-7.5.

6. The method for detecting the specific antibody in the serum of the UUR10_0579 protein by using the detection kit according to any one of claims 2 to 5, which comprises the following steps:

(1) sample adding: taking serum to be detected, adding a serum sample dilution buffer solution, and uniformly mixing;

(2) and (3) incubation: adding one drop of calf serum and one drop of serum specimen to be detected into each hole, setting two holes of negative/positive control, adding two drops of negative control or positive control into each hole, and sealing the plates for 1h at 37 ℃;

(3) washing: the microporous plate is thrown to remove liquid in the pores, washing liquid is injected, the microporous plate is placed for 10 seconds, the microporous plate is dried, and the steps are repeated for 4 times;

(4) adding an enzyme-labeled antibody: adding 1 drop of enzyme-labeled antibody into each well, and sealing the plate for 1h at 37 ℃;

(5) washing the plate as before;

(6) color development: adding one drop of each of the color developing agent A and the color developing agent B, and standing for 20min at 37 ℃;

(7) and (3) terminating the reaction: adding 1 drop of terminator into each hole;

(9) and observing and recording results: the OD optical density value at 490nm when the excitation light was read was used to calculate the antibody content.

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