CN117074668A - HPV virus typing detection kit and preparation method thereof - Google Patents
HPV virus typing detection kit and preparation method thereof Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- 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
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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Abstract
The invention belongs to the technical field of biological immunity detection analysis, and particularly relates to an HPV virus typing detection kit and a preparation method thereof. The HPV genotyping detection kit comprises a test strip and a sample extracting solution, wherein the test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorbing pad, the nitrocellulose membrane is coated with a detection line and a quality control line, the quality control line is coated with a colloidal gold-labeled mouse anti-human HPV16 protein monoclonal antibody and a mouse anti-human HPV18 protein monoclonal antibody, and the quality control line is coated with an anti-mouse HPV16IgG antibody and an HPV18IgG antibody.
Description
Technical Field
The invention belongs to the technical field of biological immunity detection analysis, and particularly relates to an HPV virus typing detection kit and a preparation method thereof.
Background
Human Papillomaviruses (HPV), belonging to the genus papovaviridae, papilloma virus a, are spherical DNA viruses that cause squamous epithelial proliferation of the human skin mucosa. 130 or more types have been isolated, and different types cause different clinical manifestations, which can be classified according to the tissue site of invasion: (1) skin low risk: including HPV1, 2, 3, 4, 7, 10, 12, 15, etc. associated with common warts, flat warts, plantar warts, etc.; (2) high risk skin: including HPV5, 8, 14, 17, 20, 36, 38, etc. are associated with epidermodysplasia verrucosa, other malignancies also associated with possible HPV infection include: vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer; (3) Mucous membrane low risk type such as HPV6, 11, 13, 32, 34, 40, 42, 43, 44, 54, etc. and infectious genitalia, anus, oropharynx, esophageal mucosa; (4) Mucosal high risk HPV16, 18, 30, 31, 33, 35, 53, 39 and cervical cancer, rectal cancer, oral cancer, tonsil cancer, etc.
There are various methods for detecting HPV, including hybridization capture method, fluorescence in situ hybridization method, chip technology hybridization method, real-time fluorescence PCR method, spot printing method, etc., and the sensitivity and specificity of each detection method are different. But can be classified into two types according to the detection result, namely quantitative detection and typing detection.
In any detection method, the requirements on equipment are high, and the detection can be performed in a laboratory with relatively perfect conditions, the average detection time is more than 4 hours, the operation is complex, and the detection method depends on a real-time quantitative PCR instrument and high-level technicians.
Disclosure of Invention
The invention aims to provide an HPV virus typing detection kit and a preparation method thereof.
According to the HPV genotyping detection kit of the specific embodiment of the invention, the kit comprises a test strip and a sample extracting solution, wherein the test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorbing pad, the nitrocellulose membrane is coated with a detection line and a quality control line, the quality control line is coated with a colloidal gold-labeled mouse anti-human HPV16 protein monoclonal antibody and a mouse anti-human HPV18 protein monoclonal antibody, and the quality control line is coated with an anti-mouse HPV16IgG antibody and an HPV18IgG antibody.
Preferably, the specimen extraction solution comprises 100mmol of Tris-HCl buffer, 25mmol of EDTA solution, 500mmol of NaCl solution and 1wt% SDS, wherein the pH of the Tris-HCl buffer is 8.0, and the pH of the EDTA solution is 8.0.
According to the HPV genotyping detection kit of the specific embodiment of the invention, an anti-mouse HPV16IgG antibody and an HPV18IgG antibody are mixed with a coating buffer solution to prepare a quality control mixed solution with the concentration of 0.5mg/mL, and the mixed solution is sprayed on a nitrocellulose membrane at the speed of coating amount of 1uL/cm to obtain a quality control line.
According to the HPV genotyping detection kit of the specific embodiment of the invention, a murine anti-human HPV16 monoclonal antibody and a coating buffer solution are mixed to prepare a mixed solution with the concentration of 0.4mg/mL, and the mixed solution is sprayed on a nitrocellulose membrane to obtain a detection line T16.
According to the HPV genotyping detection kit of the specific embodiment of the invention, a murine anti-human HPV18 monoclonal antibody is mixed with a coating buffer solution to prepare a mixed solution with the concentration of 0.4mg/mL, and the mixed solution is sprayed on a nitrocellulose membrane to obtain a detection line T18.
According to the HPV virus typing detection kit of the specific embodiment of the invention, the coating buffer solution is borate, carbonate, phosphate, tris-HCl or Tris-phosphate buffer solution, and the pH value of the buffer solution is 7.0-7.5.
The preparation method of the HPV virus typing detection kit according to the specific embodiment of the invention comprises the following steps:
(1) Adding HPV IgG antibody into gold-labeled buffer solution, adding BSA, mixing, standing to obtain precipitate, redissolving the precipitate to obtain colloidal gold-labeled HPV IgG antibody, loading on a substrate, and drying to obtain a colloidal gold pad;
(2) Coating at least two of FITC monoclonal antibody, TAMRA monoclonal antibody, DIG monoclonal antibody or CY5 monoclonal antibody on a nitrocellulose membrane, or coating at least two of FAM monoclonal antibody, TAMRA monoclonal antibody, DIG monoclonal antibody or CY5 monoclonal antibody on a nitrocellulose membrane as a detection line; coating a goat anti-mouse antibody on a nitrocellulose membrane to be used as a quality control line;
(3) The colloidal gold pad, nitrocellulose membrane, and the base plate, sample pad, and absorbent pad were assembled.
According to the HPV genotyping detection kit of the specific embodiment of the invention, in the step (2), FITC monoclonal antibody, TAMRA monoclonal antibody, DIG antibody monoclonal antibody, CY5 monoclonal antibody or mLFAM monoclonal antibody, the coating concentration of which is 0.3mg/mL, 0.2mg/mL, 0.5mg/mL and 0.3mg/mLFAM monoclonal antibody are sprayed on a nitrocellulose membrane at a speed of 1 mu L/cm to serve as detection lines. .
According to the preparation method of the HPV virus typing detection kit, a biotin monoclonal antibody is added into a gold-labeled buffer solution, a sealing solution is added, the mixture is uniformly mixed and centrifuged to obtain a precipitate, and the precipitate is redissolved to obtain the colloidal gold-labeled biotin monoclonal antibody.
According to the preparation method of the HPV virus typing detection kit, colloidal gold and 0.2M potassium carbonate are mixed to obtain the gold-labeled buffer solution, wherein gold particles of the colloidal gold are 40nm.
According to the preparation method of the HPV virus typing detection kit, the blocking solution is 10% bovine serum albumin.
The use method of the detection kit comprises the following steps:
1. sample collection:
cervical cell collection:
1. after the vaginal speculum expands the vagina, the longer brush wire in the center of the cervical brush head is inserted into the cervical canal, and the shorter brush wires at the two ends prop against the mucous membrane surface of the external orifice of the cervix until the bristles of the two wings are closely contacted with the two sides of the cervical orifice.
2. The handle is held by thumb and forefinger, and the handle rotates at least 3-5 circles in one direction (clockwise or anticlockwise) all the time by taking the cervical outer opening as the center of a circle. Proper pressure is needed to be applied during rotation. If leucorrhea is excessive, the sample should be brushed by gently wiping with sterile dry cotton.
3. Taking down the brush head by forceps or scissors, putting into the extracting solution collected by the specimen, screwing the bottle cap, shaking the brush head for a plurality of times forcefully, and taking out the brush head.
2. Sample inspection:
1. before testing, the instruction manual must be read completely and operated strictly according to the instruction manual;
2. taking out the detection card and putting the detection card flat;
3. sucking 2-3 drops of the treated sample (60-90 mu L) at one third of the depth of the liquid in the sample extracting solution from the bottom of the bottle by using a suction pipe, and vertically dripping the sample into a sample adding hole of the detection card;
4. after being placed at room temperature for 10-20 minutes, the detection result is observed
4. Interpretation of the test results:
1. positive: the two mauve strips, the detection line (T16 line) and the quality control line (C line) are both developed; or the detection line (T18 line) and the quality control line (C line) are developed;
2. negative: one purple red strip, only the quality control line (C line) develops color;
3. invalidation: the quality control line (C line) has no color development, the detection is invalid, and the detection card is recommended to be taken for re-detection.
The invention has the beneficial effects that:
the kit can directly detect the antigen of HPV virus, has higher sensibility and specificity, can rapidly carry out typing detection, does not need a PCR instrument and high-level technicians, and is simple, convenient and rapid.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows a schematic structure of a test strip in a kit of the invention, wherein the test strip comprises a 1-sample pad, a 2-binding pad, a 3-detection line T16 line, a 4-detection line T18 line, a 5-quality control line C line, a 6-absorbent pad, a 7-nitrocellulose membrane and an 8-PVC bottom plate.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1 preparation of double-labeled colloidal gold immunochromatographic test strip of the present invention
1. Preparing colloidal gold: colloidal gold prepared by trisodium citrate reduction method
50mL of 0.01% chloroauric acid solution is taken, heated and stirred until boiling, 2mL of 1% trisodium citrate solution is rapidly added after boiling, and rapid and uniform stirring is performed for 15min until the liquid becomes red and no longer changes, ultrapure water is added to 50mL after natural cooling at room temperature, and the mixture is preserved at 4 ℃ for standby.
2. Preparing gold-labeled protein:
protein to be marked: HPV IgG antibody and sheep anti-human HPV IgG antibody
Adjusting pH of colloidal gold to 7.9-8.5 with 0.1M sodium carbonate, slowly adding 5-25 g ug of protein to be marked per milliliter of colloidal gold solution, uniformly mixing, standing for 10min, then adding BSA to a final concentration of 0.5-1%, uniformly mixing, standing for 10min, centrifuging at 9000rpm for 2min, removing precipitation, transferring the upper layer solution to a new tube, centrifuging at 12500 for 10min, removing supernatant, adding heavy suspension to the original amount, transferring the solution to the new tube, centrifuging again at 12500rpm for 10min, adding a preservation solution with one tenth of the initial volume, re-suspending the precipitate, and standing at 4 ℃ for standby.
The gold-labeled proteins to be labeled include, but are not limited to, murine anti-human HPVIgG antibodies, rabbit anti-human.
3. Preparing a colloidal gold test strip:
and diluting with a coating buffer solution, detecting the protein to be coated to a concentration of 0.5-1.5mg/mL, and spraying the streptavidin and the protein to be coated on a membrane at a distance of 0.5-1.5uL/cm from a position 1cm away from the bonding pad by using a membrane-drawing gold spraying instrument to form a detection belt 4, a quality control belt 5, wherein the distance between the detection belt and the quality control belt is about 5mm, and the distance between the quality control belt and the water absorption pad is about 1cm.
The analytical membrane is dried at 37 ℃ and packaged for standby.
The coating buffer used is borate, carbonate, phosphate, tris-HCl or Tris-phosphate, etc. and the pH value of the buffer is 7.0-7.5.
4. Test strip T16 line: the murine anti-human HPV16 monoclonal antibody was mixed with 10mM PBS buffer solution at pH 7.2 to prepare a mixed solution at a concentration of 0.4mg/mL, which was sprayed onto nitrocellulose membrane at a coating weight of 1uL/cm.
5. And C line of the quality control belt: the goat anti-mouse IgG polyclonal antibody was mixed with 10m MPBS buffer solution at pH 7.5 to prepare a mixed solution with a concentration of 0.5mg/mL, and sprayed onto nitrocellulose membrane with a coating amount of 1uL/cm.
The structure of the test strip is shown in fig. 1, and the test strip comprises a sample pad (1), a bonding pad (2), a detection line T16 line (3), a detection line T18 line (4), a quality control line C line (5), a water absorption pad (6), a nitrocellulose membrane (7) and a PVC base plate (8).
The test paper strip and the card shell are assembled into a detection card, and a sample extracting solution, a sterile sampling swab, a suction pipe and a part of instruction are prepared, so that the detection kit of the invention can be completed.
Wherein, the sample extracting solution comprises the following raw materials: 100mmol Tris-HCl (pH 8.0), 25mmol EDTA (pH 8.0), 500mmol NaCl and 1% SDS.
Example 2 detection of samples
The kit prepared in this example detects clinical samples, and a total of 98 clinical samples were included. The experimental procedure included the collection and detection of samples:
and (3) collecting a sample:
1. after the vaginal speculum expands the vagina, the longer brush wire in the center of the cervical brush head is inserted into the cervical canal, and the shorter brush wires at the two ends prop against the mucous membrane surface of the external orifice of the cervix until the bristles of the two wings are closely contacted with the two sides of the cervical orifice.
2. The handle is held by thumb and forefinger, and the handle rotates at least 3-5 circles in one direction (clockwise or anticlockwise) all the time by taking the cervical outer opening as the center of a circle. Proper pressure is needed to be applied during rotation. If leucorrhea is excessive, the sample should be brushed by gently wiping with sterile dry cotton.
3. Taking down the brush head by forceps or scissors, putting into the extracting solution collected by the specimen, screwing the bottle cap, shaking the brush head for a plurality of times forcefully, and taking out the brush head.
Sample detection and result analysis:
1. before testing, the instruction manual must be read completely and operated strictly according to the instruction manual;
2. taking out the detection card and putting the detection card flat;
3. sucking 2-3 drops of the treated sample (60-90 mu L) at one third of the depth of the liquid in the sample extracting solution from the bottom of the bottle by using a suction pipe, and vertically dripping the sample into a sample adding hole of the detection card;
4. after being placed at room temperature for 10-20 minutes, the detection result is observed:
1) Positive: the two mauve strips, the detection line (T16 line) and the quality control line (C line) are both developed; or the detection line (T18 line) and the quality control line (C line) are developed;
2) Negative: one purple red strip, only the quality control line (C line) develops color;
3) Invalidation: the quality control line (C line) has no color development, the detection is invalid, and the detection card is recommended to be taken for re-detection.
Of these, 19 cases were diagnosed as positive, 79 cases were negative, 6 cases of high-risk HPV type 16 and 7 cases of high-risk HPV type 18, and the results of consistency analysis were carried out by using the detection results of the kit in this example, and the HPV-DNA detection results and the tissue immunohistochemical results of the PCR instrument, as shown in the following table:
TABLE 1 clinical sample test results
The kit of the invention is matched with the HPV-DNA method by adopting 108 samples respectively and is matched with the result of a control histological detection experiment, so that the kit of the invention can meet the clinical requirements.
Example 3 comparative experiments
(1) Conventional colloidal gold method
1.1 samples of HPV16 and HPV18 strains were simultaneously added to sample extracts of the PCR methods currently used in hospitals according to dilution criteria of 1:1000,1:500,1:200,1:100,1:500 (the components of the sample extracts are 10mol/L of tris (hydroxymethyl) aminomethane, 1mol/L of ethylenediamine tetraacetic acid; 0.02% ethylphenyl polyethylene glycol, 0.06% of sodium chloride).
Other procedure was as in example 1.
2-3 drops of the sample solution (60-90 mu L) are sucked by a suction tube and respectively vertically dripped into the sample adding holes of the detection card.
The result shows that the quality control line C corresponding to the sample solutions with different dilution ratios is fully developed, only the sample solution diluted according to the ratio of 1:50 is slightly developed when being detected T, and the rest detection lines T are not developed.
(2) The method of the invention
2.1 samples of HPV16 and HPV18 strains were added simultaneously to the sample extracts of the present invention (100 mmol Tris-HCl (pH 8.0), 25mmol EDTA (pH 8.0), 500mmol NaCl and 1% SDS) according to dilution criteria 1:1000,1:500,1:200,1:100,1:500, respectively.
Other procedure was as in example 1.
The results show that under different dilution ratios, the quality control C line and the detection T line are developed, wherein the color development of the T line of the sample card with the ratio of 1:1000 is relatively shallow.
The conventional preservation solution is mainly used for treating cell fixation, preservation and the like, and viruses are difficult to combine with antibodies in nuclei, and after the preservation solution is replaced by the preservation solution, innumerable perforations can appear on the nuclei, so that the antigen-antibody combination density is greatly improved, and the T line can still develop under different dilution concentrations.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
- The HPV genotyping detection kit is characterized by comprising a test strip and a sample extracting solution, wherein the test strip comprises a bottom plate, a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorbing pad, a detection line and a quality control line are coated on the nitrocellulose membrane, a colloidal gold-labeled mouse anti-human HPV16 protein monoclonal antibody and a mouse anti-human HPV18 protein monoclonal antibody are coated on the quality control line, and an anti-mouse HPV16IgG antibody and an HPV18IgG antibody are coated on the quality control line.
- 2. The HPV genotyping kit according to claim 1, wherein the anti-murine HPV16IgG antibody and HPV18IgG antibody are mixed with a coating buffer solution to prepare a quality control mixed solution having a concentration of 0.5mg/mL, and the mixed solution is sprayed onto a nitrocellulose membrane at a rate of 1uL/cm in coating amount to obtain a quality control line.
- 3. The HPV genotyping kit according to claim 1, wherein the murine anti-human HPV16 monoclonal antibody is mixed with the coating buffer solution to prepare a mixed solution with a concentration of 0.4mg/mL, and sprayed on a nitrocellulose membrane to obtain the detection line T16.
- 4. The HPV genotyping kit according to claim 1, wherein the murine anti-human HPV18 monoclonal antibody is mixed with the coating buffer solution to prepare a mixed solution with a concentration of 0.4mg/mL, and sprayed on a nitrocellulose membrane to obtain the detection line T18.
- 5. The HPV genotyping kit according to any one of claims 2 to 4, wherein the coating buffer is borate, carbonate, phosphate, tris-HCl or Tris-phosphate buffer, and the pH of the buffer is 7.0 to 7.5.
- 6. The method for preparing the HPV genotyping detection kit according to claim 1, wherein the method comprises the steps of:(1) Adding HPV IgG antibody into gold-labeled buffer solution, adding BSA, mixing, standing to obtain precipitate, redissolving the precipitate to obtain colloidal gold-labeled HPV IgG antibody, loading on a substrate, and drying to obtain a colloidal gold pad;(2) Coating at least two of FITC monoclonal antibody, TAMRA monoclonal antibody, DIG monoclonal antibody or CY5 monoclonal antibody on a nitrocellulose membrane, or coating at least two of FAM monoclonal antibody, TAMRA monoclonal antibody, DIG monoclonal antibody or CY5 monoclonal antibody on a nitrocellulose membrane as a detection line; coating a goat anti-mouse antibody on a nitrocellulose membrane to be used as a quality control line;(3) The colloidal gold pad, nitrocellulose membrane, and the base plate, sample pad, and absorbent pad were assembled.
- 7. The method of claim 6, wherein in step (2), FITC monoclonal antibody, TAMRA monoclonal antibody, DIG antibody monoclonal antibody, CY5 monoclonal antibody or mLFAM monoclonal antibody with a coating concentration of 0.3mg/mL, 0.2mg/mL, and 0.5mg/mL are sprayed on nitrocellulose membrane at a rate of 1. Mu.L/cm as detection lines.
- 8. The method for preparing HPV genotyping assay kit according to claim 6, wherein the biotin monoclonal antibody is added to a gold-labeled buffer, a blocking solution is added, the mixture is homogenized and centrifuged to obtain a precipitate, and the precipitate is redissolved to obtain the colloidal gold-labeled biotin monoclonal antibody.
- 9. The method for preparing HPV genotyping assay kit according to claim 6, wherein the gold-labeled buffer is obtained by mixing colloidal gold with 0.2M potassium carbonate, wherein gold particles of the colloidal gold are 40nm.
- 10. The method for preparing HPV genotyping assay kit according to claim 8 wherein the blocking solution is 10% bovine serum albumin.
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