CN112014565A - Test strip and kit for detecting trichophyton rubrum and preparation method of test strip - Google Patents
Test strip and kit for detecting trichophyton rubrum and preparation method of test strip Download PDFInfo
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- CN112014565A CN112014565A CN202011198310.0A CN202011198310A CN112014565A CN 112014565 A CN112014565 A CN 112014565A CN 202011198310 A CN202011198310 A CN 202011198310A CN 112014565 A CN112014565 A CN 112014565A
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- 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
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
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- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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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Abstract
The application discloses a test strip for detecting trichophyton rubrum, a kit and a preparation method of the test strip, and belongs to the field of microorganisms. The test strip comprises a bottom plate, and a sample pad, a marker pad, a reaction pad and a water absorption pad which are sequentially overlapped on the bottom plate, wherein the marker pad is coated with an anti-biotin antibody marked by latex particles with different colors and a specific antibody II aiming at chitin on the cell wall of trichophyton rubrum thallus; the reaction pad is provided with a detection area and a quality control area, the detection area is coated with a first specific antibody aiming at chitin on the cell wall of trichophyton rubrum thallus, and the quality control area is coated with biotin bovine serum albumin. The test strip prepared by the preparation method has strong specificity and simple and convenient operation, can obtain a detection result in a short time, eliminates the influence of subjective factors of inspectors, and can realize the rapid detection of a field sample.
Description
Technical Field
The application belongs to the field of microorganisms, and particularly relates to a test strip and a kit for detecting trichophyton rubrum, and a preparation method of the test strip.
Background
Dermatophytosis (dermatomycosis) is the infectious skin disease with the highest incidence in the population, can occur in healthy people and patients with immune insufficiency, and has extremely high recurrence rate. Since the clinical manifestations of dermatophytosis are sometimes similar to those of other skin diseases such as seborrheic dermatitis, psoriasis, candidal intertrigo, erythrasma, eczema, etc., clinical diagnosis is more difficult especially for patients with immune insufficiency.
Dermatophytes (A. sup. (B.))Dermatophytes) Is a main pathogen of dermatophytosis, is a group of filamentous parasitic fungi, mainly invades the skin, hair and nails of human beings or animals, parasitizes or saprophytoses in keratin tissues of cutin, hair and nail plate of epidermis, and causes tinea capitis, tinea corporis, tinea cruris, tinea manus, tinea pedis and onychomycosis. Dermatophytosis belongs to the family of Moniliaceae of the order of the Onospora semiperniciflua subphyla (Withania sp.). The dermatophytes can be divided into 3 genera, Trichophyton (Trichophyton) according to the characteristics of the macroconidium (Trichophyton)Trichophyton): a rod-shaped macrocarris spore; microsporophyllum genus (Microsporum): fusiform macrocarpium conidiophore; genus epidermophyton (A)Epidermophyton): pestle-like large conidia. About 45 kinds of dermatophytes have been reported, some of which only infect animals, and about 20 or more kinds of which have pathogenic effects on human. Epidemiological investigation results show that the most common dermatophytes in China and even all over the world are the trichophyton rubrum ()Trichophyton rubrum). Trichophyton rubrum is a parent dermatophyton, and can cause tinea corporis, tinea cruris, tinea manus and pedis, onychomycosis and tinea purulent, and also can cause deep infection and granuloma of dermatophyton.
At present, dermatophytes are mainly identified by morphological methods, namely direct observation under a microscope and fungus culture. However, the traditional morphological identification method is long in time consumption, and result judgment is easily influenced by external culture conditions and subjective factors of observers, so that inaccurate identification of atypical bacteria is easily caused.
Disclosure of Invention
Aiming at the problems that the identification method of the fungus morphology is long in time consumption and inaccurate in identification, the application provides a test strip and a kit for detecting trichophyton rubrum and a preparation method of the test strip.
In a first aspect, the application provides a test strip for detecting trichophyton rubrum, which adopts the following technical scheme:
a test strip for detecting trichophyton rubrum comprises a bottom plate, and a sample pad, a marker pad, a reaction pad and a water absorption pad which are sequentially overlapped on the bottom plate, wherein the marker pad is coated with an anti-biotin antibody marked by latex particles with different colors and a second specific antibody aiming at the trichophyton rubrum; the reaction pad is provided with a detection area and a quality control area, the detection area is coated with a first specific antibody aiming at trichophyton rubrum, and the quality control area is coated with biotin bovine serum albumin.
By adopting the technical scheme, the trichophyton rubrum in the sample is detected by applying the principle of the double-antibody sandwich method, the occurrence of cross reaction is reduced, the specificity is strong, the operation is simple and convenient, the detection result can be obtained in a short time, the subjective factors of inspectors are eliminated, and the rapid detection of the on-site specimen can be realized.
Specifically, the test strip of the application is internally coated with a specific antibody aiming at trichophyton rubrum in a detection area of a reaction pad, the specific antibody is used as a detection antibody, meanwhile, another specific antibody aiming at the trichophyton rubrum is combined on a marker pad and is marked by latex particles with a certain color, and the two antibodies form a set of detection system capable of developing color. Preferably, both the second specific antibody against trichophyton rubrum and the first specific antibody against trichophyton rubrum of the present application are specific antibodies against chitin on cell walls of trichophyton rubrum cells. In addition, a set of detection system which is irrelevant to the trichophyton rubrum thallus to be detected and can present another color is formed in the quality control area of the test strip reaction pad. The two systems operate independently and do not interfere with each other, so that the phenomenon that the quality control area with stronger capture capacity captures more antigens to cause false negative of a detection sample is avoided, and the double-color reaction can cause poor vision, thereby being more favorable for interpretation of a detection result.
Therefore, the test strip can be used for detecting human body specimens, namely detecting whether a subject is infected with trichophyton rubrum, can avoid the phenomenon that detection results are inaccurate due to subjective factors of detection personnel, and can effectively distinguish other skin diseases with similar clinical manifestations to the dermatophytosis, such as seborrheic dermatitis, psoriasis, candidal indirect eruption, erythrasma, eczema and the like, and is simple, convenient, rapid, objective and high in detection accuracy.
Preferably, the anti-biotin antibody is labeled with blue latex particles; the second specific antibody against trichophyton rubrum is marked by red latex particles.
Through adopting above-mentioned technical scheme, this application adopts the anti-biotin antibody of blue latex particle mark can combine and present blue with the biotin bovine serum albumin that the control area is peridium, and the specificity antibody two to the trichophyton rubrum that adopts red latex particle mark can combine and present red with the trichophyton rubrum that probably exists in the sample. So that when Trichophyton rubrum is present in the test sample, the test strip shows a blue color and a red color; when the trichophyton rubrum does not exist in the detection sample, the test strip only shows blue, and the red-blue double-color reaction causes poor vision, thereby being more beneficial to the interpretation of the detection result.
Preferably, the latex particles have a diameter of 200-500 nm. More preferably, the latex particles are 200, 250, 300, 350, 400, 450 or 500nm in diameter. Most preferably, the latex particles are 300mm in diameter.
Preferably, the latex particles are polystyrene latex particles.
Through adopting above-mentioned technical scheme, this application adopts the polystyrene latex particle of above-mentioned nanometer diameter, can carry out effective mark to the antibody to present different colours through the composite reaction, the direct observation test result of the naked eye of being convenient for. In addition, the emulsion particle of this application can select to use commercialized reagent, and the granule size is controllable, has avoided the loaded down with trivial details of experiment firing and the poor problem of batch that particle size differs and lead to, but also can shorten the preparation cycle of test paper strip, reduction in production cost.
Preferably, the sample pad is made of glass fiber material.
Through adopting above-mentioned technical scheme, the sample pad of this application is made by glass fiber, and glass fiber can reduce the loss rate of the sample that awaits measuring of dripping on the sample pad, improves the accuracy of testing result. Moreover, the glass fiber also has the advantages of higher strength, light weight, stronger corrosion resistance, especially stronger chemical corrosion resistance, and the like, and has longer service life.
Preferably, the marker pad is made of a polyester film material.
By adopting the technical scheme, the marker pad is made of the polyester film, and the polyester film (namely the PET polyester film) has low water absorption rate, so that the latex antibody conjugate can be conveniently detected and can be further chromatographed on the nitrocellulose film to carry out composite reaction. In addition, polyester films also have good mechanical strength, stiffness, hardness, good weatherability and chemical resistance, such as resistance to weak acids and organic solvents.
Preferably, the reaction pad is made of a nitrocellulose membrane material.
Through adopting above-mentioned technical scheme, the reaction pad of this application is made by nitrocellulose membrane, and nitrocellulose membrane has good chromatography performance, and has stronger protein adsorption capacity, can be wet by water under the prerequisite that does not influence protein adsorption capacity.
Preferably, the absorbent pad is made of absorbent paper.
Through adopting above-mentioned technical scheme, the paper that absorbs water is chooseed for use to the pad that absorbs water of this application, and the paper that absorbs water has good water absorption capacity, impels the detection sample to react from sample pad chromatography to reaction pad under capillary action.
Preferably, the bottom plate is made of polystyrene material.
Through adopting above-mentioned technical scheme, the bottom plate of this application is made by polystyrene, and the polystyrene material has better adhesion nature, easily combines the bottom plate with the liner material adhesion on it.
In a second aspect, the present application provides a kit for detecting trichophyton rubrum, which adopts the following technical scheme:
a kit for detecting trichophyton rubrum comprises the test strip for detecting trichophyton rubrum.
By adopting the technical scheme, the kit has high sensitivity, high detection speed, simple and convenient operation and strong objectivity, can effectively eliminate subjective factors such as the service level of an inspector, and is suitable for rapidly detecting whether trichophyton rubrum exists in a large number of samples on site.
In a third aspect, the application provides a method for preparing a test strip for detecting trichophyton rubrum, which adopts the following technical scheme:
a preparation method of the test strip for detecting trichophyton rubrum comprises the following steps:
a. the antibodies were labeled with latex particles of different colors:
s1, respectively diluting the latex particles with the two colors by using PBS solution, wherein the final concentration of the two diluted latex particles is controlled to be 0.5-5% by volume;
s2, adding an anti-biotin antibody and a second specific antibody against trichophyton rubrum into the diluted latex particles respectively, controlling the final concentration of the two antibodies to be 0.1-0.5mg/mL, and reacting at 0-4 ℃ overnight;
s3, adding BSA with the final concentration of 0.5-5 vol% into the two latex-antibody conjugates obtained in the step aS2 respectively for blocking for 0.5-4 h;
s4, respectively centrifuging the two conjugates subjected to the blocking treatment in the step aS3, removing supernatant, and re-dissolving two precipitates obtained by centrifuging by using PBS (phosphate buffer solution) containing 0.1-1 vol% BSA (bovine serum albumin) to obtain two latex-antibody conjugates;
wherein, 0.01-0.5M PBS solution with pH of 6-8 is adopted in the step aS1 and the step aS 4;
b. film dotting:
s1: respectively diluting the biotin bovine serum albumin and a specific antibody I aiming at trichophyton rubrum by using a PBS solution, wherein the concentrations of the diluted biotin bovine serum albumin and the specific antibody I are controlled to be 1-4 mg/mL;
s2, spotting the biotin bovine serum albumin diluted in the step bS1 in a quality control area of the reaction pad, spotting the specific antibody aiming at the trichophyton rubrum diluted in the step bS1 in a detection area of the reaction pad, and drying to obtain a spotted reaction pad;
wherein, PBS solution with pH6-8 of 0.01-0.5M is adopted in the step bS 1;
c. dotting latex:
s1, mixing the two latex-antibody conjugates obtained in the step a, diluting the two latex-antibody conjugates with PBS (phosphate buffer solution) containing 0.1-0.5 vol% BSA, and controlling the final concentration of the two latex-antibody conjugates in the mixed diluent to be 0.3-0.5 vol%;
s2, spraying the mixture containing the two latex-antibody conjugates obtained in the step cS1 on a marker pad, and drying to obtain the marker pad after latex spraying;
wherein, PBS solution with 0.01-0.1M pH6-8 is adopted in the step cS 1;
d. assembling: and (c) overlapping and fixing the sample pad, the marker pad obtained in the step (c), the reaction pad obtained in the step (b) and the water absorption pad on a bottom plate in sequence to obtain the test strip for detecting the trichophyton rubrum. Preferably, the sample pad, the marker pad, the reaction pad and the absorbent pad are fixed on the base plate by means of adhesion.
By adopting the technical scheme, the test strip for detecting trichophyton rubrum can be prepared quickly, conveniently and low in cost, the specificity of the prepared test strip is good, and the accuracy of a detection result is high.
Preferably, in the step bS2 and the step cS2, the drying condition is 25 to 45 ℃ for 1 to 4 hours.
By adopting the technical scheme, the marker pad and the reaction pad are dried at the temperature of 25-45 ℃, so that the stability and the activity of the antibody are improved, and the accuracy of a detection result is improved.
In summary, the present application has the following beneficial effects:
1. the test strip of the application detects trichophyton rubrum in a sample by applying the principle of a double-antibody sandwich method, reduces the occurrence of cross reaction, has strong specificity, and can effectively distinguish other skin diseases with similar clinical manifestations to dermatophytosis, such as seborrheic dermatitis, psoriasis, candidal indirect eruption, erythrasma, eczema and the like;
2. compared with a microscopic examination method, the test strip for detecting the trichophyton rubrum eliminates the influence caused by subjectivity of inspectors, and can effectively distinguish atypical bacteria; compared with a culture method, the test strip for detecting the trichophyton rubrum does not need to use instruments and equipment, is convenient to operate, has short detection time, greatly improves the detection efficiency, and can realize the rapid detection of on-site samples;
3. the two detection systems on the test strip operate independently and do not interfere with each other, so that the phenomenon that a quality control area with stronger capture capacity captures more antigens to cause false negative of a detection result is avoided, and double-color reaction can cause poor vision, so that the judgment of the detection result is facilitated;
4. the preparation method of the test strip is rapid and convenient, and the test strip adopts easily available raw materials and low price, and is suitable for large-scale production.
Drawings
Fig. 1 is a schematic structural diagram of the test strip of the present application.
Wherein, 1, a sample pad; a marker pad;
3. a reaction pad; 4, a water absorption pad;
5. a base plate; 6, detecting area;
7. and a quality control area.
Detailed Description
The present application will be described in further detail with reference to the following drawings and examples.
At present, dermatophytes are mainly identified by traditional morphological methods, namely direct observation under a microscope and fungal culture. The accuracy of the detection result of the microscopic examination method is closely related to the service capability and subjective judgment of detection personnel, and the experimental result cannot be quickly obtained on the detection site because the culture method needs instruments and equipment. Therefore, the application has creatively developed a test paper strip of trichophyton rubrum in the inspection human sample, and different with the colloidal gold mark, the development granule that the colloidal gold granule was regarded as the immunochromatography reagent is replaced to the latex particle of this application adoption different colours.
As shown in fig. 1, the test strip of the present application comprises a base plate 5, and a sample pad 1, a marker pad 2, a reaction pad 3 and a water absorption pad 4 which are sequentially overlapped and adhered on the base plate 5, wherein the marker pad 2 is coated with an anti-biotin antibody labeled with blue latex particles and a specific antibody II labeled with red latex particles and directed against chitin on the cell wall of trichophyton rubrum thallus; the reaction pad 3 is provided with a detection area 6 and a quality control area 7, the detection area 6 is coated with a specific antibody I aiming at chitin on the cell wall of trichophyton rubrum thallus, and the quality control area 7 is coated with biotin bovine serum albumin.
During sample detection, a liquid sample is dripped into a sample pad 1 made of glass fiber, liquid is chromatographed towards one end of a water absorption pad 4 made of absorbent paper due to capillary action, in the chromatography process, the liquid sample firstly redissolves a latex antibody marker on a marker pad 2 made of a polyester film, and the mixture is continuously chromatographed on a reaction pad 3 made of a nitrocellulose membrane to generate immunoreaction with antibodies of inner envelopes of a detection area 6 and a quality control area 7, so that lines with corresponding colors are shown. The specific reaction process is as follows:
1. the quality control region 7 of the nitrocellulose membrane is coated with biotin bovine serum albumin, and the polyester membrane is coated with anti-biotin antibody labeled with blue latex. After sample application, no matter whether trichophyton rubrum exists in a sample or not, the biotin bovine serum albumin can capture the anti-biotin antibody marked by the blue latex particles, and the blue color of the corresponding latex particles is displayed in a quality control area;
2. the detection area 6 of the nitrocellulose membrane is coated with a first specific antibody against the chitin on the cell wall of the trichophyton rubrum thallus, and the polyester membrane is coated with a second specific antibody against the chitin on the cell wall of the trichophyton rubrum thallus marked by red latex. After sample application, if the sample contains trichophyton rubrum, a complex of the specific antibody of trichophyton rubrum-the specific antibody of trichophyton rubrum marked by the red latex particles is formed in the detection area 6, and a red strip is formed. If the trichophyton rubrum is not contained, the compound cannot be formed, and no band appears. Thus, the presence of a red band at the detection zone 6 indicates that the sample contains Trichophyton rubrum, which is positive; otherwise, the result is negative.
The test strips of the present application were prepared using the following examples, in which the latex particles and antibodies were derived from the following sources:
the anti-biotin antibody of the present application was purchased from Changshabe Boyou Biotech, Inc., cat # AGSTV-0100;
the biotin bovine serum albumin of the present application was purchased from Changshabe good Biotech, Inc., under the product number AGBIO-0100;
the specific antibody aiming at the chitin on the cell wall of the trichophyton rubrum thallus is purchased from Changshan Boyou biological technology limited company, a product number ABCPV-0603;
the specific antibody aiming at the chitin on the cell wall of the trichophyton rubrum thallus is purchased from Changshan Boyou biological technology limited company, a product number ABCPV-0601;
the red latex particles of the present application are available from suzhou-degree biotechnology limited under the designation DR 05C;
the blue latex particles of the present application were purchased from suzhou-mindu biotechnology limited, cat # DB 05C.
Example 1
A preparation method of the test strip for detecting trichophyton rubrum comprises the following steps:
a. the antibodies were labeled with latex particles of different colors:
(1) blue latex particle labeled anti-biotin antibody
Diluting 300nm blue latex particles to a final concentration of 1 vol% with 0.25M PBS solution at pH 7.4; adding an anti-biotin antibody to the blue latex particle solution to a final concentration of 0.25mg/mL, and reacting at 2 ℃ overnight; adding BSA with the final concentration of 1 vol% into the latex antibody conjugate for blocking for 2 h; the mixture was centrifuged at 13000 rpm/min for 13min, the supernatant was removed, and the precipitate was reconstituted with 0.25M PBS pH7.4 containing 0.5 vol% BSA to give a latex-antibody conjugate. Measuring the absorbance at 500nm, wherein the concentration is 1 vol%, and standing at 2 deg.C for use;
(2) specific antibody II marked by red latex particles and aiming at chitin on cell walls of trichophyton rubrum thalli
The preparation method is the same as that of the anti-biotin antibody marked by blue latex particles;
b. film dotting:
respectively diluting the biotin bovine serum albumin and a specific antibody I aiming at chitin on the cell wall of trichophyton rubrum thallus by using 0.25M PBS (phosphate buffer solution) with pH7.4, wherein the concentration of the diluted biotin bovine serum albumin and the diluted specific antibody I is 2 mg/mL; respectively putting diluted biotin bovine serum albumin and specific antibody aiming at chitin on trichophyton rubrum thallus cell wall on a quality control area 7 and a detection area 6 of a reaction pad 3 by using a film dotting machine, drying for 2 hours at 37 ℃ to obtain the reaction pad 3 subjected to film dotting, sealing and placing at room temperature for later use;
c. dotting latex:
mixing the two latex-antibody conjugates obtained in step a and diluting with 0.25M PBS (pH7.4) containing 0.25 vol% BSA, wherein the final concentration of each latex-antibody conjugate after dilution is controlled to be 0.4 vol%; spraying a mixture containing two latex-antibody conjugates on the marker pad 2 by a machine, drying for 2 hours at 37 ℃ to obtain the marker pad 2 with the latex, and sealing and placing at room temperature for later use;
d. assembling: and (4) overlapping and sticking the sample pad 1, the marker pad 2 obtained in the step (c), the reaction pad 3 obtained in the step (b) and the water absorption pad 4 on a bottom plate 5 in sequence, cutting into corresponding widths, and packaging to obtain the test strip for detecting trichophyton rubrum.
e. Sample detection:
s1, preparing a specimen treatment solution: tween was diluted with 0.01M PBS pH7.4 to a final concentration of 0.5 vol%;
s2, testing: 10 drops (about 500. mu.L) of the specimen treatment liquid was dropped vertically into the specimen-processing tube, and then the specimen was added into the specimen-processing tube, the dropper of the specimen-processing tube was closed, and the above-mentioned liquid specimen was added to the sample pad 1. If the specimen is nail, skin or hair, the specimen is placed in the specimen processing tube and then the bottom of the specimen processing tube is continuously extruded for about 1 minute; if the sample is a cotton swab, the sample is placed into the sample processing pipe, then the sample processing pipe at the head of the cotton swab needs to be extruded, the cotton swab is rotated for 15 times, then the cotton swab is placed in the sample processing pipe for standing for 2 minutes, the cotton swab is extruded to extrude liquid in the cotton swab as much as possible, and finally the cotton swab is discarded.
f. And (4) interpretation of results:
if the specimen contains trichophyton rubrum, a red latex-marked specific trichophyton rubrum antibody-compound is formed in the detection area 6, and a red strip appears; if the specimen does not contain Trichophyton rubrum, the compound cannot be formed, and no band appears.
Whether the sample contains trichophyton rubrum or not, the biotin-BSA in the quality control region 7 can capture the anti-biotin antibody labeled with blue latex, so that a blue band appears.
g. And (3) clinical detection:
the test paper prepared in example 1 and a conventional microscopic method were used for detecting 300 human specimens.
The conventional microscopic examination method comprises the following steps:
1. directly collecting a specimen from a human body and preparing the specimen by a potassium hydroxide method: placing the collected specimen in the center of an objective, dripping 1 drop of potassium hydroxide to seal the solid and liquid, covering a cover plate, slightly heating above the flame of an alcohol lamp to a degree of not boiling, placing for several minutes, slightly pressing the cover plate, and sucking off the redundant liquid by using a cotton swab, so that microscopic examination can be carried out;
2. and (3) observing under a microscope: when in microscopic examination, hypha, spore or thallus in the specimen is found by using a low-power microscope, and then verified by using a high-power microscope, so that a report can be issued;
3.10% -40% of potassium hydroxide sealing liquid: typically, a concentration of 15% is used, for example, in examining specimens with thicker cutin, the concentration of potassium hydroxide can be increased to 20% -40%.
The formula is as follows: 10g-40g of potassium hydroxide (AR)
Distilled water to 100ml
After the potassium hydroxide is completely dissolved, shaking up and storing in a glass bottle.
Note that: because the concentration of potassium hydroxide is high and the smear is corrosive, the smear is easy to dry and form potassium hydroxide crystals, which affects the observation and can not be stored for a long time.
4. And (4) interpretation of results: usually, hyphae are separated and connected with the nodal chain. The microconidia is tear drop type, and is distributed along hypha individually, and the large conidia can be formed at the end of the large hypha directly, individually or in clusters. Typically characterized by the fact that the microconidia are produced directly on the macrocconidia, the articular conidia are easily produced from hyphae and macrocconidia.
The results of the experiment are shown in Table 1.
The sensitivity, specificity and accuracy of the test strips of example 1 were analyzed and calculated using a data induction Table (Square Table) for evaluation of test authenticity, see Table 2:
sensitivity = [ a/(a + c) ]. times.100%
Specificity = [ d/(b + d) ]. times.100%
Accuracy = [ (a + d)/(a + b + c + d) ] × 100%
By calculation, compared with the conventional microscopic examination method, the test strip prepared in the embodiment 1 of the application has 92.79% of sensitivity, 98.41% of specificity and 96.33% of accuracy on clinical human body specimens. The experimental result shows that the test strip prepared in the embodiment 1 can be used for detecting trichophyton rubrum in a human body sample, so that a good foundation is laid for realizing the rapid detection of a field specimen.
Example 2
A preparation method of the test strip for detecting trichophyton rubrum comprises the following steps:
a. the antibodies were labeled with latex particles of different colors:
(1) blue latex particle labeled anti-biotin antibody
200nm blue latex particles were diluted to a final concentration of 0.5 vol% with 0.01M PBS solution pH 6; adding an anti-biotin antibody to the blue latex particle solution to a final concentration of 0.1mg/mL, and reacting at 0 ℃ overnight; adding BSA with a final concentration of 0.5 vol% to the latex antibody conjugate for blocking for 0.5 h; the mixture was centrifuged at 12000 rpm/min for 15min, the supernatant was removed, and the precipitate was reconstituted with 0.01 pH6 PBS containing 0.1 vol% BSA to give a latex-antibody conjugate. Measuring absorbance at 500nm to give a concentration of 0.8 vol%, and standing at 0 deg.C;
(2) specific antibody II marked by red latex particles and aiming at chitin on cell walls of trichophyton rubrum thalli
The preparation method is the same as that of the preparation method of the anti-biotin antibody marked by the blue latex particles except that the red latex particles with the wavelength of 500nm are adopted;
b. film dotting:
respectively diluting the biotin bovine serum albumin and a specific antibody I aiming at chitin on cell walls of trichophyton rubrum thalli by using 0.01M PBS (phosphate buffer solution) with pH6, wherein the diluted concentrations of the biotin bovine serum albumin and the specific antibody I are both 1 mg/mL; respectively putting diluted biotin bovine serum albumin and specific antibody aiming at chitin on trichophyton rubrum thallus cell wall on a quality control area 7 and a detection area 6 of a reaction pad 3 by using a film dotting machine, drying for 4 hours at 25 ℃ to obtain the reaction pad 3 subjected to film dotting, sealing and placing at room temperature for later use;
c. dotting latex:
mixing the two latex-antibody conjugates obtained in the step a, diluting the mixture by using 0.01M PBS (phosphate buffer solution) with pH6 containing 0.1 vol% BSA, wherein the final concentrations of the diluted antibiotic antibody marked by blue latex particles and the specific antibody II marked by red latex particles and aiming at the chitin on the cell wall of trichophyton rubrum are respectively 0.3 vol% and 0.5 vol%; spraying a mixture containing two latex-antibody conjugates on the marker pad 2 by a machine, drying for 4 hours at 25 ℃ to obtain the marker pad 2 with the latex, and sealing and placing at room temperature for later use;
d. assembling: and (4) overlapping and sticking the sample pad 1, the marker pad 2 obtained in the step (c), the reaction pad 3 obtained in the step (b) and the water absorption pad 4 on a bottom plate 5 in sequence, cutting into corresponding widths, and packaging to obtain the test strip for detecting trichophyton rubrum.
e. Sample detection and result interpretation: same as example 1;
f. and (3) clinical detection:
299 human body samples are detected by the test strip prepared in the embodiment 2 and a conventional microscopic examination method. The conventional microscopic examination method was the same as the method described in example 1. The results of the experiment are shown in Table 3.
Sensitivity, specificity and accuracy data of the test strip prepared in example 2 were obtained by the analytical calculation method described in example 1. By calculation, compared with the conventional microscopic examination method, the test strip prepared in the embodiment 2 of the application has the sensitivity of 95.00% to clinical human body specimens, the specificity of 98.99% and the accuracy of 97.66%. The experimental result shows that the test strip prepared in the embodiment 2 can be used for detecting trichophyton rubrum in a human body sample, so that a good foundation is laid for realizing the rapid detection of a field specimen.
Example 3
A preparation method of the test strip for detecting trichophyton rubrum comprises the following steps:
a. the antibodies were labeled with latex particles of different colors:
(1) blue latex particle labeled anti-biotin antibody
The 400nm blue latex particles were diluted to a final concentration of 5 vol% with 0.5M PBS solution at pH 8; adding an anti-biotin antibody to the blue latex particle solution to a final concentration of 0.5mg/mL, and reacting at 4 ℃ overnight; adding BSA with a final concentration of 5 vol% to the latex antibody conjugate for blocking for 4 h; the mixture was centrifuged at 13000 rpm/min for 10min, the supernatant removed, and the pellet redissolved in 0.5M PBS pH8 containing 1% BSA by volume to give a latex-antibody conjugate. Measuring absorbance at 500nm with concentration of 1.2 vol%, and standing at 4 deg.C;
(2) specific antibody II marked by red latex particles and aiming at chitin on cell walls of trichophyton rubrum thalli
Except that red latex particles with the particle size of 350nm are adopted, the preparation method is the same as that of the preparation method of the anti-biotin antibody marked by the blue latex particles;
b. film dotting:
respectively diluting the biotin bovine serum albumin and a specific antibody I aiming at chitin on the cell wall of trichophyton rubrum thallus by using 0.5M PBS (phosphate buffer solution) with pH8, wherein the diluted concentrations of the biotin bovine serum albumin and the specific antibody I are both 4 mg/mL; respectively putting diluted biotin bovine serum albumin and specific antibody aiming at chitin on trichophyton rubrum thallus cell wall on a quality control area 7 and a detection area 6 of a reaction pad 3 by using a film dropping machine, drying for 1 hour at 45 ℃ to obtain the reaction pad 3 after film dropping, sealing and placing at room temperature for later use;
c. dotting latex:
mixing the two latex-antibody conjugates obtained in the step a, diluting the mixture by using 0.1M PBS (phosphate buffer solution) with pH8 containing 0.5 vol% BSA, wherein the final concentrations of the diluted antibiotic antibody marked by blue latex particles and the specific antibody II marked by red latex particles and aiming at the chitin on the cell wall of trichophyton rubrum are respectively 0.5 vol% and 0.3 vol%; spraying a mixture containing two latex-antibody conjugates on the marker pad 2 by a machine, drying for 1 hour at 45 ℃ to obtain the marker pad 2 with the latex, and sealing and placing at room temperature for later use;
d. assembling: and (4) overlapping and sticking the sample pad 1, the marker pad 2 obtained in the step (c), the reaction pad 3 obtained in the step (b) and the water absorption pad 4 on a bottom plate 5 in sequence, cutting into corresponding widths, and packaging to obtain the test strip for detecting trichophyton rubrum.
e. Sample detection and result interpretation: same as example 1;
f. and (3) clinical detection:
331 human specimens were tested by the test strip prepared in example 3 and the conventional microscopy method. The conventional microscopic examination method was the same as the method described in example 1. The results are shown in Table 4.
Sensitivity, specificity and accuracy data of the test strip prepared in example 3 were obtained by the analytical calculation method described in example 1. By calculation, compared with the conventional microscopic examination method, the test strip prepared in the embodiment 3 of the application has the sensitivity of 94.96% to clinical human body specimens, the specificity of 97.40% and the accuracy of 96.37%. The experimental result shows that the test strip prepared in the embodiment 3 can be used for detecting trichophyton rubrum in a human body sample, so that a good foundation is laid for realizing the rapid detection of a field specimen.
The embodiments of the present invention are preferred embodiments of the present application, and the scope of protection of the present application is not limited by the embodiments, so: all equivalent changes made according to the structure, shape and principle of the present application shall be covered by the protection scope of the present application.
Claims (10)
1. The utility model provides a detect test paper strip of trichophyton rubrum, includes bottom plate (5) and on bottom plate (5) overlap joint's sample pad (1), marker pad (2), reaction pad (3) and pad (4) absorb water in proper order, its characterized in that: the marker pad (2) is coated with an anti-biotin antibody which is marked by latex particles with different colors and a second specific antibody aiming at trichophyton rubrum; a detection area (6) and a quality control area (7) are formed on the reaction pad (3), the detection area (6) is coated with a first specific antibody aiming at trichophyton rubrum, and the quality control area (7) is coated with bovine serum albumin biotin.
2. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the anti-biotin antibody is marked by blue latex particles; the second specific antibody against trichophyton rubrum is marked by red latex particles.
3. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the diameter of the latex particles is 200-500 nm.
4. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the sample pad (1) is made of glass fiber materials.
5. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the marker pad (2) is made of polyester film material.
6. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the reaction pad (3) is made of a nitrocellulose membrane material.
7. The test strip for detecting trichophyton rubrum according to claim 1, wherein: the bottom plate (5) is made of polystyrene material.
8. A kit for detecting trichophyton rubrum is characterized in that: comprising the test strip for detecting trichophyton rubrum according to any one of claims 1 to 7.
9. A method for preparing the test strip for detecting trichophyton rubrum of any one of claims 1 to 7, wherein the method comprises the steps of: the method comprises the following steps:
a. the antibodies were labeled with latex particles of different colors:
s1, respectively diluting the latex particles with the two colors by using PBS solution, wherein the final concentration of the two diluted latex particles is controlled to be 0.5-5% by volume;
s2, respectively adding an anti-biotin antibody and a second specific antibody aiming at trichophyton rubrum thallus into the diluted latex particles, controlling the final concentration of the two antibodies to be 0.1-0.5mg/mL, and reacting at 0-4 ℃ overnight;
s3, adding BSA with the final concentration of 0.5-5 vol% into the two latex-antibody conjugates obtained in the step aS2 respectively for blocking for 0.5-4 h;
s4, respectively centrifuging the two conjugates subjected to the blocking treatment in the step aS3, removing supernatant, and re-dissolving two precipitates obtained by centrifuging by using PBS (phosphate buffer solution) containing 0.1-1 vol% BSA (bovine serum albumin) to obtain two latex-antibody conjugates;
b. film dotting:
s1: respectively diluting the biotin bovine serum albumin and a specific antibody I aiming at trichophyton rubrum by using a PBS solution, wherein the concentrations of the diluted biotin bovine serum albumin and the specific antibody I are controlled to be 1-4 mg/mL;
s2, spotting the biotin bovine serum albumin diluted in the step bS1 on a quality control area (7) of the reaction pad (3), spotting the specific antibody aiming at the trichophyton rubrum diluted in the step bS1 on a detection area (6) of the reaction pad (3), and drying to obtain the spotted reaction pad (3);
c. dotting latex:
s1, mixing the two latex-antibody conjugates obtained in the step a, and diluting the mixture by using PBS (phosphate buffer solution) containing 0.1-0.5 vol% BSA, wherein the final concentration of the two latex-antibody conjugates after dilution is controlled to be 0.3-0.5 vol%;
s2, spraying the mixture containing the two latex-antibody conjugates obtained in the step cS1 on the label pad (2), and drying to obtain the latex-coated label pad (2);
d. assembling: and (c) overlapping and fixing the sample pad (1), the marker pad (2) obtained in the step (c), the reaction pad (3) obtained in the step (b) and the water absorption pad (4) on a bottom plate (5) in sequence to obtain the test strip for detecting trichophyton rubrum.
10. The method for preparing the test strip for detecting trichophyton rubrum according to claim 9, wherein the test strip comprises: in the step bS2 and the step cS2, the drying is carried out at 25 to 45 ℃ for 1 to 4 hours.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113820484A (en) * | 2021-08-27 | 2021-12-21 | 中国医学科学院皮肤病医院(中国医学科学院皮肤病研究所) | Preparation method of household test strip for rapidly detecting dermatophytes |
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