阅读说明：本技术 一种同时检测滴虫、加德纳菌、白色念珠菌的量子点荧光免疫层析试纸及其制备方法 (Quantum dot fluorescence immunochromatography test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and preparation method thereof ) 是由 贺然 张小爽 马溶 马丽雅 陈国柱 黄琼 漆新国 于 2021-08-20 设计创作，主要内容包括：本发明提供了一种同时检测滴虫、加德纳菌、白色念珠菌的量子点荧光免疫层析试纸及其制备方法,属于体外诊断试剂技术领域。本发明提供的一种同时检测滴虫、加德纳菌、白色念珠菌的高灵敏量子点荧光免疫层析试纸及其制备方法,基于双抗体夹心法检测原理进行检测,对灵敏度高度提升,提高疾病检测结果准确性；实现对三种病原微生物滴虫、加德纳菌和白色念珠菌的同时检测,操作简单、快速,5～10min获得检测结果；实现一次加样即可完成三项检测,样本用量低,50～80μl样本即可完成三项检测,具有较大的市场应用价值。(The invention provides a quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and a preparation method thereof, belonging to the technical field of in vitro diagnostic reagents. The high-sensitivity quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and the preparation method thereof provided by the invention have the advantages that the detection is carried out based on the double-antibody sandwich method detection principle, the sensitivity is highly improved, and the accuracy of a disease detection result is improved; the method has the advantages that the simultaneous detection of three pathogenic microorganisms, namely trichomonas, gardnerella and candida albicans is realized, the operation is simple and rapid, and the detection result is obtained within 5-10 min; three detections can be completed by one-time sample adding, the sample consumption is low, and the three detections can be completed by 50-80 mul of sample, so that the method has a great market application value.)

1. A quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans comprises a bottom plate, and a sample pad, a filter pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered to the bottom plate from bottom to top, and is characterized in that 1 quality control line C and 3 detection lines T are arranged on the nitrocellulose membrane, and the 3 detection lines T are respectively coated with a goat anti-trichomonas antibody, a goat anti-gardnerella antibody and a goat anti-candida albicans antibody; the quality control line C is coated with anti-sheep IgG; the combined pad is sprayed with sheep anti-trichomonas antibody marked quantum dot microspheres, sheep anti-gardnerella antibody marked quantum dot microspheres and sheep anti-white antibody marked quantum dot microspheres.

2. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 1, which is characterized in that the preparation method of the goat anti-trichomonas antibody labeled quantum dot microsphere, the goat anti-gardnerella antibody labeled quantum dot microsphere or the goat anti-candida albicans antibody labeled quantum dot microsphere comprises the following steps:

1) sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the washed quantum dot microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activated quantum dot microsphere solution;

2) diluting the activated quantum dot microsphere solution, dropwise adding a sheep anti-trichomonas antibody solution, a sheep anti-gardnerella antibody solution or a sheep anti-candida albicans antibody solution for coupling reaction, and obtaining the sheep anti-trichomonas antibody marked quantum dot microsphere solution, the sheep anti-gardnerella antibody marked quantum dot microsphere solution and the sheep anti-candida albicans antibody marked quantum dot microsphere solution through sealing, separation, precipitation and re-dissolution.

3. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 2, wherein the mass concentration of the quantum dot microsphere solution is 0.8-1.2%;

the final adding concentration of the N-hydroxysuccinimide or the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is independently 1-5 mg/ml;

the time of the activation reaction is 25-35 min.

4. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 2, wherein the dilution factor of the activated quantum dot microsphere solution in the step 2) is 10-100 times.

5. The quantum dot fluorescence immunochromatographic test strip for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 4, wherein the volume of the activated quantum dot microsphere solution and the mass ratio of the goat anti-trichomonas antibody, the goat anti-gardnerella antibody or the goat anti-candida albicans antibody are independently 1 ml: 2-5 μ g;

the concentration of the sheep anti-trichomonas antibody solution, the sheep anti-gardnerella antibody solution or the sheep anti-candida albicans antibody solution is 0.01-0.1 mg/ml;

the dropping speed is 1-5 s/drop; the volume of each dropping liquid is 30-50 mu l.

6. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to any one of claims 2 to 4, characterized in that the goat anti-trichomonas antibody labeled quantum dot microspheres, the goat anti-gardnerella antibody labeled quantum dot microspheres and the goat anti-white antibody labeled quantum dot microspheres are mixed according to the volume ratio of 1:1:1, and the obtained mixed solution is sprayed on the binding pad, wherein the spraying amount is 0.1 to 3 μ l/cm.

7. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 1, wherein the coating concentration of the anti-sheep IgG is 1-2 mg/ml;

the coating concentration of the sheep anti-trichomonas antibody, the sheep anti-gardnerella antibody or the sheep anti-candida albicans antibody is 0.1-3 mg/ml independently.

8. The quantum dot fluorescence immunochromatographic test strip for simultaneously detecting trichomonas, gardnerella and candida albicans according to claim 1 or 7, wherein the goat anti-trichomonas antibody, goat anti-gardnerella antibody or goat anti-candida albicans antibody is prepared from a mouse immunized with a trichomonas antigen, a gardnerella antigen or a candida albicans antigen.

9. The quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans according to any one of claims 1 to 5 and 7, which is characterized in that the sample pad is obtained by soaking the sample pad in a treatment solution and then drying the sample pad;

the treatment solution is PBS buffer solution with 9-11 mM pH7.3-7.5, which contains 0.09-0.11% of BSA (bovine serum albumin), 9-11% of sucrose and 0.09-0.11% of Tween-20 by volume.

10. The method for preparing the quantum dot fluorescence immunochromatographic test paper for simultaneously detecting the trichomonas, gardnerella and candida albicans according to any one of claims 1 to 9, which is characterized by comprising the following steps;

and sticking the nitrocellulose membrane, the filter pad, the combination pad, the sample pad and the absorbent paper on the bottom plate, and cutting into strips.

Technical Field

The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to quantum dot fluorescence immunochromatography test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and a preparation method thereof.

Background

Trichomonas vaginalis (Trichomonas vaginalis) is a flagellate which parasitizes in human vagina and urinary tract, mainly causes Trichomonas vaginitis and urethritis, and is an infectious disease mainly transmitted by sexuality.

Gardnerella vaginalis (Gardnerella varenalis) is a gram-negative or positive brevibacterium, and is anaerobic, hemophilous and difficult to culture. Is one of the main pathogenic bacteria of female bacterial vaginosis, can be transmitted by sexual contact, and can cause poor pregnancy fate such as oviduct pregnancy, premature rupture of fetal membranes, premature birth of newborn and the like.

Candida albicans (Candida albicans) is in an oval shape, and germinates from geminispores and cells to elongate to form pseudohyphae, and the pseudohyphae are connected with the spores to form branches or chains. 80-90% of mycotic vaginal infections are caused by Candida albicans.

The mainstream technology of the existing trichomonas, gardnerella and candida albicans detection technology is a microscope observation method, the accuracy of the detection result is mainly determined by the subjective judgment of an inspection doctor, and the individual difference is obvious and the objectivity is poor. Although the existing detection products developed by the colloidal gold immunochromatographic technology solve the problem of convenience, the colloidal gold has poor sensitivity and low coincidence rate with clinical diagnosis results. In addition, the problem of sample size is also involved, and currently, vaginal sub-sampling is basically adopted, so that the sample size is small, and the detection of a plurality of items is not supported sufficiently on the premise of ensuring the detection concentration.

Disclosure of Invention

In view of the above, the present invention aims to provide a quantum dot fluorescence immunochromatographic test strip for simultaneously detecting trichomonas, gardnerella and candida albicans and a preparation method thereof, wherein the test strip has a small demand for a detection sample, and has high sensitivity and detection result accuracy.

The invention provides a quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans, which comprises a bottom plate, and a sample pad, a filter pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered to the bottom plate from bottom to top, wherein the nitrocellulose membrane is provided with 1 quality control line C and 3 detection lines T, and the 3 detection lines T are respectively coated with a goat anti-trichomonas antibody, a goat anti-gardnerella antibody and a goat anti-candida albicans antibody; the quality control line C is coated with anti-sheep IgG; the combined pad is sprayed with sheep anti-trichomonas antibody marked quantum dot microspheres, sheep anti-gardnerella antibody marked quantum dot microspheres and sheep anti-white antibody marked quantum dot microspheres.

Preferably, the preparation method of the goat anti-trichomonas antibody marked quantum dot microsphere, the goat anti-gardnerella antibody marked quantum dot microsphere or the goat anti-candida albicans antibody marked quantum dot microsphere comprises the following steps:

1) sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the washed quantum dot microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activated quantum dot microsphere solution;

2) diluting the activated quantum dot microsphere solution, dropwise adding a sheep anti-trichomonas antibody solution, a sheep anti-gardnerella antibody solution or a sheep anti-candida albicans antibody solution for coupling reaction, and obtaining the sheep anti-trichomonas antibody marked quantum dot microsphere solution, the sheep anti-gardnerella antibody marked quantum dot microsphere solution and the sheep anti-candida albicans antibody marked quantum dot microsphere solution through sealing, separation, precipitation and re-dissolution.

Preferably, the mass concentration of the quantum dot microsphere solution is 0.8-1.2%;

the final adding concentration of the N-hydroxysuccinimide or the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is independently 1-5 mg/ml;

the time of the activation reaction is 25-35 min.

Preferably, the dilution multiple of the activated quantum dot microsphere solution in the step 2) is 10-100 times.

Preferably, the volume of the activated quantum dot microsphere solution and the mass ratio of the sheep anti-trichomonas antibody, the sheep anti-gardnerella antibody or the sheep anti-candida albicans antibody are 1 ml: 2-5 μ g;

the concentration of the sheep anti-trichomonas antibody solution, the sheep anti-gardnerella antibody solution or the sheep anti-candida albicans antibody solution is 0.01-0.1 mg/ml independently;

the dropping speed is 1-5 s/drop; the volume of each dropping liquid is 30-50 mu l.

Preferably, the goat anti-trichomonas antibody-labeled quantum dot microspheres, the goat anti-gardnerella antibody-labeled quantum dot microspheres and the goat anti-white antibody-labeled quantum dot microspheres are mixed according to the volume ratio of 1:1:1, and the obtained mixed solution is sprayed on the bonding pad, wherein the spraying amount is 0.1-3 mul/cm;

preferably, the coating concentration of the anti-sheep IgG is 1-2 mg/ml;

the coating concentration of the sheep anti-trichomonas antibody, the sheep anti-gardnerella antibody or the sheep anti-candida albicans antibody is 0.1-3 mg/ml independently.

Preferably, the goat anti-trichomonas antibody, goat anti-gardnerella antibody or goat anti-candida albicans antibody is prepared by immunizing a mouse with a trichomonas antigen, a gardnerella antigen or a candida albicans antigen.

Preferably, the sample pad is obtained by soaking the sample pad in a treatment solution and then drying the sample pad;

the treatment solution is 9-11 mM PBS buffer solution with pH of 7.3-7.5, which contains 0.09-0.11% of BSA (bovine serum albumin), 9-11% of sucrose and 0.09-0.11% of Tween-20 by volume.

The invention provides a preparation method of the quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans, which comprises the following steps;

and sticking the nitrocellulose membrane, the filter pad, the combination pad, the sample pad and the absorbent paper on the bottom plate, and cutting into strips.

The invention provides a quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans, wherein a nitrocellulose membrane is provided with 1 quality control line C and 3 detection lines T, and the 3 detection lines T are respectively coated with a goat anti-trichomonas antibody, a goat anti-gardnerella antibody and a goat anti-candida albicans antibody; the quality control line C is coated with anti-sheep IgG; the combined pad is sprayed with sheep anti-trichomonas antibody marked quantum dot microspheres, sheep anti-gardnerella antibody marked quantum dot microspheres and sheep anti-white antibody marked quantum dot microspheres. The test paper is used for detecting by adopting a sandwich method principle, the simultaneous detection of three pathogenic microorganisms, namely trichomonas, gardnerella and candida albicans, is simple to operate, can finish three detections by one-time sample adding, has less demand on samples, can finish three detections by 50-80 mu l of liquid samples, and has high detection efficiency. Compared with the conventional product, the test paper is rapid in detection, and a detection result is obtained in 5-10 min; in addition, the sensitivity of the test paper is greatly improved compared with that of a colloidal gold methodology.

Drawings

Fig. 1 is a schematic structural diagram of the test strip provided by the present invention, wherein 1, detection lines T1, 2, detection lines T2, 3, detection lines T3, 4, quality control lines C, 5, and sample wells.

Detailed Description

The invention provides a quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans, which comprises a bottom plate, and a sample pad, a filter pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially adhered to the bottom plate from bottom to top, wherein the nitrocellulose membrane is provided with 1 quality control line C and 3 detection lines T, and the 3 detection lines T are respectively coated with a goat anti-trichomonas antibody, a goat anti-gardnerella antibody and a goat anti-candida albicans antibody; the quality control line C is coated with anti-sheep IgG; the combined pad is sprayed with sheep anti-trichomonas antibody marked quantum dot microspheres, sheep anti-gardnerella antibody marked quantum dot microspheres and sheep anti-white antibody marked quantum dot microspheres.

In the present invention, the test strip includes a conjugate pad. The combined pad is sprayed with goat anti-trichomonas antibody marked quantum dot microspheres, goat anti-gardnerella antibody marked quantum dot microspheres and goat anti-white antibody marked quantum dot microsphere solution. The goat anti-trichomonas antibody marked quantum dot microsphere solution, the goat anti-gardnerella antibody marked quantum dot microsphere solution and the goat anti-white antibody marked quantum dot microsphere solution are mixed according to the volume ratio of 1:1:1, and the obtained mixed solution is sprayed on the bonding pad. The spraying amount of the mixed solution is preferably 0.1-3 mul/cm, more preferably 0.5-2.5 mul/cm, and most preferably 2 mul/cm. The concentrations of the goat anti-trichomonas antibody marked quantum dot microsphere solution, the goat anti-gardnerella antibody marked quantum dot microsphere solution and the goat anti-white antibody marked quantum dot microsphere solution are independent, preferably 0.01-0.1 mu M, more preferably 0.02-0.08 mu M, further preferably 0.04-0.07 mu M, and most preferably 0.05 mu M.

In the invention, the preparation method of the goat anti-trichomonas antibody marked quantum dot microsphere solution, the goat anti-gardnerella antibody marked quantum dot microsphere solution or the goat anti-candida albicans antibody marked quantum dot microsphere solution comprises the following steps:

1) sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the washed quantum dot microsphere solution for activation reaction, separating the precipitate, washing and dispersing to obtain an activated quantum dot microsphere solution;

2) diluting the activated quantum dot microsphere solution, dropwise adding a sheep anti-trichomonas antibody solution, a sheep anti-gardnerella antibody solution or a sheep anti-candida albicans antibody solution for coupling reaction, and obtaining the sheep anti-trichomonas antibody marked quantum dot microsphere solution, the sheep anti-gardnerella antibody marked quantum dot microsphere solution and the sheep anti-candida albicans antibody marked quantum dot microsphere solution through sealing, separation, precipitation and re-dissolution.

The method comprises the steps of sequentially adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to a washed quantum dot microsphere solution for activation reaction, separating precipitates, washing and dispersing to obtain the activated quantum dot microsphere solution.

In the present invention, the mass concentration of the quantum dot microsphere solution is preferably 0.8% to 1.2%, and more preferably 1%. The particle size of the quantum dot microsphere is preferably 100-500 nm, more preferably 150-450 nm, further preferably 200-400 nm, and further preferably 300 nm. The source of the quantum dot microspheres is not particularly limited in the invention, and quantum dot microspheres well known in the field can be adopted. In the present example, the quantum dot microspheres were purchased from siemer feishel technologies (china) ltd. The washing method is preferably to centrifuge the quantum dot microsphere solution, discard the supernatant, resuspend the solution to the original volume by using morpholine ethanesulfonic acid (MES) buffer solution, and obtain the washed quantum dot microsphere solution after ultrasonic treatment. The centrifugal force of the centrifugal machine is preferably 7500-8500 g, and more preferably 8000 g. The time for centrifugation is preferably 8-12 min, and most preferably 10 min. The concentration of the morpholine ethanesulfonic acid buffer solution is preferably 10 mM. The power of the ultrasonic wave is preferably 150-200W, and more preferably 180W. The time of the ultrasonic treatment is preferably 0.1-1 min, and more preferably 0.5 min.

In the present invention, the final concentration of the N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide added is independently preferably 1 to 5mg/ml, more preferably 1.5 to 4.5mg/ml, even more preferably 2 to 4mg/ml, and most preferably 3 mg/ml. The N-hydroxysuccinimide or 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is preferably formulated with 10mM pH 6 MES buffer, at an initial concentration of greater than 20 mg/ml. The time of the activation reaction is preferably 25-35 min, and more preferably 30 min. The temperature of the activation reaction is preferably carried out under room temperature conditions. The method for separating the precipitate is preferably performed by centrifugation. The centrifugal force of the centrifugal machine is preferably 7500-8500 g, and more preferably 8000 g. The time for centrifugation is preferably 8-12 min, and most preferably 10 min. And preferably, after washing, dispersing, namely, resuspending and redissolving the precipitate obtained by centrifugation to the original volume by using 10mM pH 6 MES buffer solution, and performing ultrasonic homogenization to form an activated quantum dot microsphere solution.

After obtaining the activated quantum dot microsphere solution, diluting the activated quantum dot microsphere solution, dropwise adding a sheep anti-trichomonas antibody solution, a sheep anti-gardnerella antibody solution or a sheep anti-candida albicans antibody solution for coupling reaction, and obtaining the sheep anti-trichomonas antibody marked quantum dot microsphere solution, the sheep anti-gardnerella antibody marked quantum dot microsphere solution and the sheep anti-candida albicans antibody marked quantum dot microsphere solution through sealing, separation, precipitation and re-dissolution.

In the invention, the dilution multiple of the activated quantum dot microsphere solution is preferably 10-100 times, more preferably 20-80 times, further preferably 40-70 times, and most preferably 50 times. The volume of the activated quantum dot microsphere solution and the mass ratio of the sheep anti-trichomonas antibody, the sheep anti-gardnerella antibody or the sheep anti-candida albicans antibody are preferably 1 ml: 2-5 μ g, more preferably 1 ml: 3-4 μ g, most preferably 1 ml: 3.5. mu.g. The concentration of the sheep anti-trichomonas antibody solution, the sheep anti-gardnerella antibody solution or the sheep anti-candida albicans antibody solution is 0.01-0.1 mg/ml independently, more preferably 0.02-0.08 mg/ml, most preferably 0.04-0.6 mg/ml, and most preferably 0.05 mg/ml. In the embodiment of the invention, the goat anti-trichomonas antibody, the goat anti-gardnerella antibody or the goat anti-candida albicans antibody are all obtained by entrusting the development of Beijing Davingji Biotechnology Co., Ltd and belong to polyclonal antibodies. The dripping speed is preferably 1-5 s/drop, more preferably 2-4 s/drop and most preferably 3 s/drop. The volume of each drop is preferably 30 to 50. mu.l, and more preferably 40. mu.l. The coupling reaction lasts for preferably 1-4 h, more preferably 2-3 h, and most preferably 2.5 h. The method of blocking is not particularly limited in the present invention, and a blocking method known in the art may be used. In the embodiment of the present invention, the blocking is preferably performed by adding 20% BSA solution to the coupling reaction solution to a final concentration of 0.1% -0.2%. The closing time is preferably 0.5-1 h, and more preferably 0.7-0.9 h. The separation and precipitation are preferably carried out by centrifugation. The preferable re-dissolution is performed by using 10mM PBS buffer solution with pH7.4 containing 0.1-1% BSA, 5-10% sucrose and 0.1-1% Tween-20. The volume of the redissolution is preferably up to the volume of the quantum dot microsphere solution before the reaction. The material of the conjugate pad preferably comprises a fiberglass film or a polyester film.

In the invention, the test paper comprises a nitrocellulose membrane. The nitrocellulose membrane is provided with 1 quality control line C and 3 detection lines T, and the quality control line C is arranged above the 3 detection lines T. The 3 detection lines T are respectively coated with a goat anti-trichomonas antibody, a goat anti-gardnerella antibody and a goat anti-candida albicans antibody. The quality control line C is coated with anti-sheep IgG. The coating concentration of the anti-sheep IgG is preferably 1-2 mg/ml, and more preferably 1.5 mg/ml. The coating concentration of the goat anti-trichomonas antibody, the goat anti-gardnerella antibody or the goat anti-candida albicans antibody is preferably 0.1-3 mg/ml independently, more preferably 0.5-2.5 mg/ml, further preferably 0.6-2 mg/ml, and most preferably 0.8 mg/ml. The goat anti-trichomonas antibody, the goat anti-gardnerella antibody or the goat anti-candida albicans antibody are obtained by development of Beijing Davingji biotechnology limited and belong to polyclonal antibodies.

In the present invention, the test strip includes a sample pad. The sample pad is obtained by drying after the treatment liquid, preferably soaking. The treatment solution is PBS buffer solution with 9-11 mM of pH 7.3-7.5, which contains 0.09-0.11% of BSA, 9-11% of sucrose and 0.09-0.11% of Tween-20 by volume, and more preferably PBS buffer solution with 10mM of pH7.4, which contains 0.1% of BSA, 10% of sucrose and 0.1% of Tween-20 by volume.

In the invention, the test paper comprises a filter pad. The filter pad is preferably a hydrophilic material, such as a glass fiber or polyethersulfone filter pad. The filter pad can realize effective interception of particles larger than 0.5 mu m.

In the present invention, the test paper preferably further includes a card case. The test paper is placed in a clamping shell and sealed for storage.

The invention provides a preparation method of the quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans, which comprises the following steps;

and sequentially sticking the sample pad, the filter pad, the combination pad, the nitrocellulose membrane and the absorbent paper on the bottom plate from bottom to top, and cutting into strips.

In the invention, the cutting width of the test paper is preferably 3-5 mm, and more preferably 3.5 mm. And when the test paper card comprises a card shell, the cut test paper is put into the card shell to form the test paper card.

The detection method of the quantum dot immunochromatographic test paper is completed based on a sandwich method, when one or more antigens of trichomonas, gardnerella and candida albicans exist in a sample, the trichomonas, gardnerella or candida albicans move upwards along with water flow under a chromatography acting force to reach a binding pad and are bound with quantum dot microspheres marked by corresponding antibodies to form a binary compound, when the binary compound moves upwards along with the chromatography acting force to reach a nitrocellulose membrane, the antigens in the binary compound are bound with the corresponding antibodies to form a ternary compound, the antibodies are fixedly coated on the nitrocellulose membrane, the ternary compound is fixed on a detection line T, meanwhile, the redundant quantum dot microspheres marked by the antibodies on the binding pad reach a quality control line C of the nitrocellulose membrane along with the chromatography acting force and perform specific affinity reaction with anti-sheep IgG to form the binary compound, the method for generating a fluorescence signal under the detection of a detector comprises the following specific operation method:

1) dripping a sample to be detected on a sample pad of the test paper, standing for 5-10 min, reading fluorescent signals of a quality control line C and a detection line T by using an ultraviolet lamp, and judging that the sample to be detected is positive and contains an antigen coated with an antibody by the corresponding detection line T when the quality control line C and the detection line T simultaneously display the fluorescent signals; when only the quality control line C displays a fluorescent signal and the detection line T does not display the fluorescent signal, judging that the sample to be detected is a negative sample and does not contain a corresponding detection object or the concentration of the detection object contained in the sample is lower than the detection limit of the test paper; when only the detection line T displays a fluorescence signal and the quality control line C does not display the fluorescence signal, the detection is wrong, and the sample needs to be detected again. The experimental result shows that the detection result of the detection test paper prepared by the invention is consistent with that of a colloidal gold method, the detection result can be obtained within 10-15 minutes, the detection sensitivity is obviously improved, and the operation is very simple.

The present invention provides a quantum dot fluorescence immunochromatographic test strip for simultaneously detecting trichomonas, gardnerella and candida albicans and a preparation method thereof, which are described in detail below with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A high-sensitivity quantum dot fluorescence immunochromatographic test paper for simultaneously detecting trichomonas, gardnerella and candida albicans and a preparation method thereof are disclosed, and the preparation method comprises the following steps:

taking 90 mul of quantum dot microspheres with the particle size of 200nm and the concentration of 1 mul, centrifuging for 10 minutes at 8000g, carefully sucking and discarding supernatant, re-suspending and re-dissolving the precipitate to 90 mul by using 10mM pH 6 MES buffer solution, and uniformly performing ultrasonic treatment for later use;

respectively preparing an NHS solution and an EDC solution with the concentration of 50mg/ml by using a 10mM pH 6 MES buffer solution, adding 5 mu l of NHS into the microspheres, uniformly mixing, then adding 5 mu l of EDC solution, uniformly mixing, reacting for 30 minutes, centrifuging for 10 minutes at 8000g, carefully sucking and discarding supernatant, re-suspending the precipitate by using a 10mM pH 6 MES buffer solution to 90 mu l, and uniformly performing ultrasonic treatment to form the activated quantum dot microspheres. Dividing the activated quantum dot microspheres into 3 pieces, each of which is 30 mu l for standby;

respectively diluting 0.1mg of goat anti-trichomonas antibody, goat anti-gardner antibody and goat anti-candida albicans antibody to 0.01mg/ml by using 10mM PBS buffer solution with pH7.4, taking a 30 microliter of activated quantum dot microsphere, diluting to 2ml by using 10mM PBS buffer solution with pH7.4, transferring to a clean beaker, putting into a clean rotor, dropwise adding all 0.01mg/ml of goat anti-trichomonas antibody, goat anti-gardner antibody or goat anti-candida albicans antibody while stirring, reacting for 2 hours after dropwise addition, then adding 20% of BSA0.1ml for sealing, reacting for 1 hour, transferring the solution to a 30ml centrifuge tube, centrifuging for 45 minutes at 8000g, and discarding the supernatant; re-dissolving the precipitate to 300 μ l with 10mM PBS buffer (pH7.4) containing 0.1% BSA, 10% sucrose and 0.1% Tween-20 to form goat anti-trichomonas antibody-labeled quantum dot microspheres, goat anti-Gardner antibody-labeled quantum dot microspheres and goat anti-Candida albicans antibody-labeled quantum dot microspheres;

respectively mixing the goat anti-trichomonas antibody marked quantum dot microspheres, the goat anti-gardner antibody marked quantum dot microspheres and the goat anti-candida albicans antibody marked quantum dot microspheres in a volume ratio of 1:1:1, spraying the mixed quantum dot microspheres onto a polyester fiber film by a film spraying instrument according to the film spraying amount of 2 mul/cm, and drying to form the quantum dot conjugate pad.

Respectively diluting the goat anti-trichomonas antibody, the goat anti-gardner antibody, the goat anti-candida albicans antibody and the anti-goat IgG to 1mg/ml by using 10mM PBS buffer solution with pH7.4, respectively preparing T1, T2, T3 and a quality control line C coating solution, coating the coating solution on a nitrocellulose membrane with the climbing speed of 135sec/4cm by using a spraying and scratching instrument according to the scratching amount of 1 mul/cm, and drying for 4 hours for later use;

immersing a blank glass fiber membrane into 10mM PBS (phosphate buffer solution) with pH7.4 containing 0.1% BSA, 10% sucrose and 0.1% Tween-20, taking out and drying after the glass fiber membrane is completely immersed, and preparing a sample pad;

and sequentially sticking the nitrocellulose membrane, the quantum dot conjugate pad, the filter pad, the sample pad and the absorbent paper on the bottom plate, cutting the nitrocellulose membrane into 3.8mm specifications, and putting the nitrocellulose membrane, the quantum dot conjugate pad, the filter pad, the sample pad and the absorbent paper into a card shell to finish the preparation of the test strip.

Example 2

Detection method

Diluting 10 times of the trichomonas positive sample, the Gardner positive sample and the Candida albicans positive sample by using normal saline, dripping 70 mu l of each gradient sample into a sample adding hole of the detection card, reacting for 10 minutes, judging a result under the irradiation of an ultraviolet lamp, and judging the positive by emitting a fluorescence band by the corresponding detection line. Simultaneously detecting latex chromatography detection card (purchased from Beijing Taige science and technology Limited company, product name is Candida albicans/Trichomonas vaginalis/Gardnerella vaginalis antigen combined detection kit (latex chromatography))

The results are shown in Table 1 and Table 3.

TABLE 1 statistical table of the test results of two test methods on positive trichomonas samples

Dilution factor of positive trichomonas sample	1：1	1：10	1：100	1：1000	1：10000
						Test results of the inventive reagent	Positive for	Positive for	Negative of	Negative of	Negative of
Colloidal gold test results	Weak positive	Negative of	Negative of	Negative of	Negative of

TABLE 2 statistical table of the test results of two test methods on Gardner's bacteria positive samples

Rare Gardner bacteria positive sampleMultiple of release	1：1	1：10	1：100	1：1000	1：10000
						Test results of the inventive reagent	Positive for	Positive for	Positive for	Positive for	Negative of
Colloidal gold test results	Positive for	Weak positive	Negative of	Negative of	Negative of

TABLE 3 statistical table of the test results of two test methods on Gardner's bacteria positive samples

The test result shows that the sensitivity of the test strip is greatly improved compared with that of a colloidal gold methodology, the detection omission phenomenon caused by sampling factors, sample concentration factors and the like can be effectively avoided, and non-specific false positive results are not found in the detection process.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.