A kit for detecting treponema pallidum and hepatitis c virus antibody in oral mucosa exudate based on colloidal selenium method

Abstract

The present application relates to the technical field of biomedical detection, and particularly relates to a kit for detecting treponema pallidum and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method, and a test strip for detecting treponema pallidum and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method. The test strip comprises a base plate, a sample pad, a colloidal selenium pad, a nitrocellulose membrane and a water absorption pad. The sample pad, the colloidal selenium pad, the nitrocellulose membrane and the water absorption pad are sequentially and adjacently pasted on the base plate in the chromatographic direction. The nitrocellulose membrane is provided with a quality control line and two detection lines. The quality control line is coated with sheep anti-mouse IgG. The two detection lines are respectively coated with TP recombinant antigen 2 and HCV recombinant antigen 2. The colloidal selenium pad is coated with colloidal selenium-labeled SPA. The kit disclosed by the present application has the advantages of convenient use and simple operation. The detection sample is oral mucosa exudate, and the non-invasive sampling avoids cross infection caused by blood sampling and other use modes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical detection, in particular to a kit for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method. BACKGROUND

[0002] Syphilis is caused by Treponema pallidum subsp. pallidum, also known as Treponema pallidum, which infects the human body and causes a chronic, systemic sexually transmitted disease. It can cause damage to multiple systems and organs of the human body, produce various clinical manifestations, lead to tissue destruction, dysfunction, and even endanger life.

[0003] Hepatitis C, also known as hepatitis C and hepatitis C, is a viral hepatitis caused by hepatitis C virus infection. It is mainly transmitted through blood transfusion, needle puncture, etc. Hepatitis C is a global epidemic and can cause chronic inflammation and fibrosis of the liver. Some patients can develop cirrhosis and even hepatocellular carcinoma.

[0004] Studies have shown that antibodies can be detected in the urine, oral mucosa exudate, semen, and vaginal secretions of syphilis and hepatitis C patients. Oral mucosa exudate and blood are both parts of human body fluids, and their components are mostly similar. The target substances appear consistently in oral mucosa exudate and blood during clinical detection, with only a difference in concentration. These findings provide a new direction for the detection of syphilis and hepatitis C.

[0005] Currently available patents in China for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate can only detect a single indicator, and all are prepared using colloidal gold methodology. Due to the size limitation of colloidal gold particles used as carriers, the sensitivity of colloidal gold method is generally low, and the produced kits cannot meet the market demand.

[0006] Therefore, it is urgent to develop a kit for simultaneously detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate. SUMMARY

[0007] In view of the shortcomings of the prior art, the present application provides a kit for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method, which has the advantages of easy use, simple operation, and easy popularization. The detection sample is oral mucosa exudate, which is non-invasive sampling, avoiding cross-infection caused by blood sampling and other use methods.

[0008] In a first aspect, the present application provides a test strip for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method, which adopts the following technical solution:

[0009] The test strip for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method comprises a base plate, a sample pad, a colloidal selenium pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the colloidal selenium pad, the nitrocellulose membrane and the water absorption pad are sequentially and adjacently pasted on the base plate in the chromatographic direction, the nitrocellulose membrane is provided with a quality control line and two detection lines, the quality control line is coated with sheep anti-mouse IgG, and the two detection lines are respectively coated with TP recombinant antigen 2 and HCV recombinant antigen 2, and the colloidal selenium pad is coated with colloidal selenium-labeled SPA.

[0010] The present application qualitatively detects free syphilis spirochetes (TP) antibodies and hepatitis C virus (HCV) antibodies in human oral mucosa exudate by adopting an indirect method and a double-antigen sandwich principle.

[0011] When the sample contains TP / HCV antibodies during detection, the TP / HCV antibodies in the sample react with the colloidal selenium-labeled SPA on the binding pad to form a labeled antigen-antibody complex, the complex is chromatographed upwards, is captured by the detection line (TP recombinant antigen 2 / HCV recombinant antigen 2) coated on the nitrocellulose membrane, and a red band appears. The complex continues to be chromatographed upwards, is captured by the quality control line (C line) antibody (sheep anti-mouse IgG) coated on the nitrocellulose membrane, and a red band appears; when the content of the analyte in the sample is lower than the minimum detection limit, the detection line does not develop color.

[0012] Preferably, the preparation method of the colloidal selenium-labeled SPA is as follows:

[0013] (1) potassium carbonate is added to the colloidal selenium solution and mixed, SPA and DNP-BSA are added and mixed, and then a blocking solution is added for reaction to obtain colloidal selenium conjugate A;

[0014] (2) potassium carbonate is added to the colloidal selenium solution and mixed, DNP is added and mixed, and then a blocking solution is added for reaction to obtain colloidal selenium conjugate B;

[0015] (3) colloidal selenium conjugate A and colloidal selenium conjugate B are mixed at a volume ratio of 1:(0.5-2) to obtain colloidal selenium-labeled SPA.

[0016] Preferably, the addition amount of the colloidal selenium solution in step (1) is 8-12 mL; the addition amount of potassium carbonate is 70-100 μL, and the addition amount of the blocking solution is 0.5-1 mL.

[0017] Preferably, the addition amount of SPA in step (1) is 200-300 μg, for example, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg.

[0018] Preferably, the amount of DNP-BSA added in step (1) is 200-300 μg, for example 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg.

[0019] Preferably, the mixing time of SPA and DNP-BSA in step (1) is 10-20 min; the reaction time of blocking solution is 10-20 min.

[0020] Preferably, the amount of colloidal selenium solution added in step (2) is 8-12 mL; the amount of potassium carbonate added is 70-100 μL; the amount of blocking solution added is 0.5-1 mL.

[0021] Preferably, the amount of DNP added in step (2) is 200-300 μg; for example 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg or 300 μg.

[0022] Preferably, the mixing time of DNP in step (2) is 10-20 min; the reaction time of blocking solution is 10-20 min.

[0023] In the present application, the DNP-BSA added in colloidal selenium conjugate A is a deoxyribonucleoprotein, and the DNP added in colloidal selenium conjugate B is a deoxyribonucleoprotein antibody; there is specific binding between DNP-BSA and DNP, and neither of them will cause non-specific adsorption with other proteins; colloidal selenium conjugate A (SPA-colloidal selenium-DNP) is mixed with colloidal selenium conjugate B (colloidal selenium-DNP-BSA), and through the specific binding of DNP-BSA and DNP, a structure of colloidal selenium-DNP-BSA-DNP-colloidal selenium-SPA is formed, so that the number of colloidal selenium particles coupled with a single SPA protein is increased, and when specific capture occurs at the detection line, more colloidal selenium particles are aggregated at the detection line, thereby enhancing the positive color development and improving the sensitivity of the product.

[0024] In the present application, DNP-BSA is added in the SPA link labeled with colloidal selenium, and DNP labeled with colloidal selenium alone, which plays a signal amplification effect.

[0025] In a specific embodiment, a method for preparing a kit for detecting Treponema pallidum and hepatitis C virus antibodies in oral mucosal exudate based on colloidal selenium method, comprising the following steps:

[0026] (1) Nitrocellulose membrane preparation: TP recombinant antigen 2, HCV recombinant antigen 2, and goat anti-mouse IgG were diluted to 0.5-1.2 mg / mL with coating solution, and were coated on the corresponding positions of the nitrocellulose membrane in turn using a membrane marker, and were dried at 45°C for 2 hours; wherein the coating solution has the following formula: 2.4 g / L Tris (pH 8.0), 20 g / L trehalose.

[0027] (2) Preparation of colloidal selenium conjugate (colloidal selenium-labeled SPA)

[0028] Preparation method of colloidal selenium conjugate A: 8-12 mL of colloidal selenium solution was taken in a beaker, 70-100 μL of 0.2M potassium carbonate solution was added and mixed, then 200-300 μg of SPA and 200-300 μg of DNP-BSA were added, and the mixture was stirred at room temperature for 10-20 min; 0.5-1 mL of 10% BSA was added and blocked for 10-20 min, centrifuged (temperature 4°C, speed 8000-10000 rpm, 15 min) to discard the supernatant, and 1 mL of storage solution was added for reconstitution, to obtain colloidal selenium conjugate A; wherein the storage solution has the following formula: 2.4 g / L Tris (pH 8.0), 100-200 g / L sucrose, 5-10 g / L BSA, and 5-10 g / L PEG20000.

[0029] Preparation method of colloidal selenium conjugate B: 8-12 mL of colloidal selenium solution was taken in a beaker, 70-100 μL of 0.2M potassium carbonate solution was added and mixed, then 200-300 μg of DNP was added, and the mixture was stirred at room temperature for 10-20 min; 0.5-1 mL of 10% BSA was added and blocked for 10-20 min, centrifuged (temperature 4°C, speed 8000-10000 rpm, 15 min) to discard the supernatant, and 1 mL of storage solution was added for reconstitution, to obtain colloidal selenium conjugate B; wherein the storage solution has the following formula: 2.4 g / L Tris (pH 8.0), 100-200 g / L sucrose, 5-10 g / L BSA, and 5-10 g / L PEG20000.

[0030] The colloidal selenium conjugate A and colloidal selenium conjugate B were mixed in a volume ratio of 1:(0.5-2) to obtain the colloidal selenium conjugate.

[0031] (3) Preparation method of colloidal selenium pad: the colloidal selenium-labeled conjugate (colloidal selenium-labeled SPA) was diluted 20-40 times and soaked into a blank pad, and the pad was taken out and dried at 37°C to obtain the colloidal selenium pad.

[0032] (4) Sample pad treatment: The blank pad is soaked in the sample pad treatment solution, and is taken out and dried at 37°C. The sample pad treatment solution formula used is: 12-24 g / L Tris (pH 8.0), 5 g / L casein, 5 g / L Tween-20, 5 g / L surfactant S7, and 2.5 g / L EDTA;

[0033] (5) Test strip assembly: The sample pad, colloidal selenium pad, nitrocellulose membrane, and water absorption pad are sequentially and sequentially spliced and pasted on the PVC bottom plate in the chromatographic direction, and the test strip is obtained after cutting according to the width requirement.

[0034] Preferably, the preparation process of the colloidal selenium solution is as follows:

[0035] S1, mixing cetyltrimethylammonium bromide and polyvinylpyrrolidone 40000;

[0036] S2, after mixing with selenous acid, mixing with ascorbic acid to obtain a colloidal selenium solution.

[0037] Preferably, the amount of cetyltrimethylammonium bromide added in step S1 is 0.8-1.2 g; for example, 0.8 g, 0.9 g, 1 g, 1.1 g, or 1.2 g.

[0038] Preferably, the amount of polyvinylpyrrolidone 40000 added in step S1 is 0.8-1.2 g; for example, 0.8 g, 0.9 g, 1 g, 1.1 g, or 1.2 g.

[0039] Preferably, in step S2, the amount of selenous acid added is 0.8-1.2 mL; and the amount of ascorbic acid added is 5-10 mL.

[0040] Preferably, in step S1, the reaction time is 8-15 min; and in step S2, the time for mixing with ascorbic acid is 8-15 min.

[0041] In this application, the colloidal selenium particles prepared by using cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone 40000 (PVP40) as templates are more uniform and stable, which helps to improve the sensitivity and specificity of the product.

[0042] In a specific embodiment, the preparation process of the colloidal selenium solution is as follows:

[0043] (1) Weigh 0.8-1.2 g of CTAB and 0.8-1.2 g of PVP40 into a beaker, add 100 mL of purified water, and stir at room temperature for 8-15 min;

[0044] (2) Add 0.8-1.2 mL of selenous acid with a concentration of 128 g / L, and stir to mix at room temperature;

[0045] (3) add 5-10 mL of ascorbic acid with a concentration of 176 g / L, stir the reaction at room temperature for 8-15 min to obtain a colloidal selenium solution.

[0046] In a second aspect, the application provides a kit, which uses the following technical solution:

[0047] A kit comprising the test strip described above.

[0048] In summary, the application includes at least one of the following beneficial technical effects:

[0049] 1. The application discloses a kit for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method, which can detect oral mucosa exudate samples without invasive sampling, thereby avoiding cross infection caused by blood sampling and other use methods.

[0050] 2. The application discloses a kit for detecting syphilis spirochetes and hepatitis C virus antibodies in oral mucosa exudate based on colloidal selenium method, which uses colloidal selenium as a carrier, is convenient and fast to detect, can complete result interpretation without the aid of related instruments and equipment, and has higher sensitivity than colloidal gold. DETAILED DESCRIPTION

[0051] The raw materials involved in the application are all commercially available products, including:

[0052] Sheep anti-mouse IgG is purchased from: Oucheng Biotechnology (Shandong) Co., Ltd., product model: A102-Ab1;

[0053] TP recombinant antigen 2 is purchased from: Jiangsu Shenji Biological Technology Co., Ltd., product model: TP recombinant antigen II;

[0054] HCV recombinant antigen 2 is purchased from: Jiangsu Shenji Biological Technology Co., Ltd., product model: HCV recombinant antigen II;

[0055] SPA is purchased from: Hangzhou Xianzhi Biological Technology Co., Ltd., product model: Protein A.

[0056] The abbreviations and key terms involved in this embodiment are defined as follows:

[0057] SPA: Staphylococcus aureus protein A;

[0058] DNP-BSA: deoxyribonucleoprotein;

[0059] DNP: deoxyribonucleoprotein antibody;

[0060] EDTA: disodium ethylenediaminetetraacetate;

[0061] BSA: bovine serum albumin;

[0062] Casein: casein;

[0063] Tris: Tris(hydroxymethyl)aminomethane;

[0064] CTAB: cetyltrimethylammonium bromide;

[0065] PVP40: polyvinylpyrrolidone 40000;

[0066] PEG20000: polyethylene glycol 20000;

[0067] SDS: sodium dodecyl sulfate;

[0068] PVA: polyethylene glycol.

[0069] The application will be further described in detail below in combination with examples and comparative examples.

[0070] Example 1: A kit for detecting Treponema pallidum and hepatitis C virus in oral mucosa exudate based on colloidal selenium method

[0071] 1. Solution preparation: 0.2M potassium carbonate preparation: 2.76g of potassium carbonate was placed in a beaker, 100mL of purified water was added, and after stirring and mixing, it was stored at 4°C for standby.

[0072] 10% BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added, and after stirring and mixing, it was stored at 4°C for standby.

[0073] Coating liquid preparation: 2.4g of Tris, 20g of trehalose were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8.0 with HCL solution, and it was stored at 4°C for standby.

[0074] Preservative solution preparation: 2.4g of Tris, 10g of BSA, 100g of sucrose, 10g of PEG20000 were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8.0 with HCL solution, and it was stored at 4°C for standby.

[0075] Sample pad treatment solution preparation: 24g of Tris, 5g of Casein, 5g of Tween-20, 5g of surfactant S7, 2.5g of EDTA were weighed into a beaker, 1L of purified water was added, and after stirring and mixing, the pH was adjusted to 8.0 with HCL solution, and it was stored at 4°C for standby.

[0076] 2. Colloidal selenium preparation

[0077] (1) 1g of CTAB and 1g of PVP40 were weighed into a beaker, 100mL of purified water was added, and stirred at room temperature for 10min;

[0078] (2) Add 1 mL of 128 g / L selenite and stir at room temperature to mix well;

[0079] (3) Add 5 mL of 176 g / L ascorbic acid and stir at room temperature for 10 min to obtain colloidal selenium solution.

[0080] 3. Preparation of nitrocellulose membranes:

[0081] The TP recombinant antigen 2 was diluted to 1 mg / mL with coating solution and coated on the detection line T1 using a coating instrument at a parameter of 1 μL / cm.

[0082] The HCV recombinant antigen 2 was diluted to 1 mg / mL with coating solution and coated on the T2 position of the detection line using a coating instrument at a parameter of 1 μL / cm.

[0083] Goat anti-mouse IgG was diluted to 1 mg / mL with coating buffer and coated at the control line position using a coating instrument at a parameter of 1 μL / cm. The coated nitrocellulose membrane was placed in a 45℃ oven and dried for 2 hours.

[0084] 4. Preparation method of colloidal selenium couplings:

[0085] Preparation method of colloidal selenium coupling compound A: Take 10 mL of colloidal selenium solution in a beaker, add 85 μL of 0.2 M potassium carbonate solution and mix well, then add 250 μg SPA and 250 μg DNP-BSA, stir and react at room temperature for 10 min; add 0.5 mL of 10% BSA to block for 10 min, centrifuge (temperature 4℃, speed 8500 rpm, 15 min), discard the supernatant, add 1 mL of preservation solution to reconstitute, and obtain colloidal selenium coupling compound A;

[0086] Preparation method of colloidal selenium conjugate B: Take 10 mL of colloidal selenium solution in a beaker, add 85 μL of 0.2 M potassium carbonate solution and mix well, then add 250 μg of DNP and stir at room temperature for 10 min; add 0.5 mL of 10% BSA to block for 10 min, centrifuge (temperature 4℃, speed 8500 rpm, 15 min), discard the supernatant, add 1 mL of preservation solution to reconstitute, and obtain colloidal selenium conjugate B; mix colloidal selenium conjugate A and colloidal selenium conjugate B at a volume ratio of 1:1 to obtain colloidal selenium conjugate (i.e., colloidal selenium-labeled SPA).

[0087] 5. Preparation method of colloidal selenium pads:

[0088] The colloidal selenium-labeled conjugate (i.e., colloidal selenium-labeled SPA) was diluted 30 times with the preservation solution and then used to soak blank pads. After being removed and dried at 37°C, the colloidal selenium pads were obtained.

[0089] 6. Sample pad treatment: Immerse the blank pad in the sample pad treatment solution, then remove and dry at 37°C.

[0090] 7. Test strip assembly: The sample pad, colloidal selenium pad, nitrocellulose membrane and water absorption pad are sequentially and sequentially bonded on the PVC base plate in the chromatographic direction, and cut into test strips with a width of 3 mm.

[0091] Example 2: A kit for detecting syphilis spirochetes and hepatitis C virus in oral mucosal exudate based on colloidal selenium method

[0092] 1. Solution preparation: 0.2M potassium carbonate preparation: 2.76g of potassium carbonate was added to a beaker with 100mL of purified water, stirred and mixed, and then stored at 4°C for later use.

[0093] 10% BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added, stirred and mixed, and then stored at 4°C for later use.

[0094] Coating solution preparation: 2.4g of Tris, 20g of trehalose were weighed into a beaker, 1L of purified water was added, stirred and mixed, and then the pH was adjusted to 8.0 with HCL solution, and stored at 4°C for later use.

[0095] Preservation solution preparation: 2.4g of Tris, 5g of BSA, 100g of sucrose, 5g of PEG20000 were weighed into a beaker, 1L of purified water was added, stirred and mixed, and then the pH was adjusted to 8.0 with HCL solution, and stored at 4°C for later use.

[0096] Sample pad treatment solution preparation: 12g of Tris, 5g of Casein, 5g of Tween-20, 5g of surfactant S7, 2.5g of EDTA were weighed into a beaker, 1L of purified water was added, stirred and mixed, and then the pH was adjusted to 8.0 with HCL solution, and stored at 4°C for later use.

[0097] 2. Colloidal selenium preparation

[0098] (1) 1g of CTAB and 0.8g of PVP40 were weighed into a beaker, 100mL of purified water was added, and stirred at room temperature for 10min;

[0099] (2) 1mL of selenous acid with a concentration of 128g / L was added, and stirred at room temperature until mixed;

[0100] (3) 10mL of ascorbic acid with a concentration of 176g / L was added, and stirred at room temperature for 10min to obtain a colloidal selenium solution.

[0101] 3. Preparation of nitrocellulose membrane:

[0102] The TP recombinant antigen 2 was diluted with the coating solution to 0.8mg / mL, and coated on the detection line T1 position with a membrane marker at a parameter of 1μL / cm;

[0103] Dilute HCV recombinant antigen 2 with coating solution to 0.8 mg / mL, coat on the position of test line T2 with a film marking instrument at a parameter of 1 μL / cm;

[0104] Dilute goat anti-mouse IgG with coating solution to 1 mg / mL, coat on the position of quality control line with a film marking instrument at a parameter of 1 μL / cm; place the coated nitrocellulose membrane in an oven at 45℃ and dry for 2 hours.

[0105] 4. Preparation method of colloidal selenium conjugate:

[0106] Preparation method of colloidal selenium conjugate A: take 10 mL colloidal selenium solution in a beaker, add 70 μL 0.2M potassium carbonate solution, mix well, then add 200 μg SPA and 200 μg DNP-BSA, stir at room temperature for 10 min; add 0.5 mL 10% BSA and block for 10 min, centrifuge (temperature 4℃, speed 8500 rpm, 15 min) to discard the supernatant, add 1 mL storage solution to reconstitute, to obtain colloidal selenium conjugate A;

[0107] Preparation method of colloidal selenium conjugate B: take 10 mL colloidal selenium solution in a beaker, add 70 μL 0.2M potassium carbonate solution, mix well, then add 200 μg DNP, stir at room temperature for 10 min; add 0.5 mL 10% BSA and block for 10 min, centrifuge (temperature 4℃, speed 8500 rpm, 15 min) to discard the supernatant, add 1 mL storage solution to reconstitute, to obtain colloidal selenium conjugate B; mix colloidal selenium conjugate A and colloidal selenium conjugate B according to a volume ratio of 1:0.5, to obtain colloidal selenium conjugate (i.e. colloidal selenium-labeled SPA).

[0108] 5. Preparation method of colloidal selenium pad:

[0109] Dilute the colloidal selenium-labeled conjugate (i.e. colloidal selenium-labeled SPA) with storage solution by 20 times, then soak the blank pad, take out and dry at 37℃ to obtain colloidal selenium pad.

[0110] 6. Treatment of sample pad: soak the blank pad in sample pad treatment solution, take out and dry at 37℃.

[0111] 7. Assembly of test strip: sequentially and sequentially joint and paste the sample pad, colloidal selenium pad, nitrocellulose membrane and water absorption pad on the PVC bottom plate in the chromatographic direction, cut into a test strip with a width of 3 mm.

[0112] Example 3: A kit for detecting Treponema pallidum and hepatitis C virus in oral mucosa exudate based on colloidal selenium method

[0113] 1. Preparation of solution: 0.2M potassium carbonate solution: 2.76g potassium carbonate in a beaker, add 100 mL purified water, mix well after stirring, and store at 4℃ for standby use.

[0114] 10% BSA preparation: weigh 10 g BSA in a beaker, add 100 mL purified water, stir and mix well, and store at 4°C for later use.

[0115] Coating solution preparation: weigh 2.4 g Tris, 20 g trehalose in a beaker, add 1 L purified water, stir and mix well, and adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0116] Preservation solution preparation: weigh 2.4 g Tris, 10 g BSA, 100 g sucrose, 7.5 g PEG20000 in a beaker, add 1 L purified water, stir and mix well, and adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0117] Sample pad treatment solution preparation: weigh 18 g Tris, 5 g Casein, 5 g Tween-20, 5 g surfactant S7, 2.5 g EDTA in a beaker, add 1 L purified water, stir and mix well, and adjust the pH to 8.0 with HCL solution, and store at 4°C for later use.

[0118] 2. Preparation of colloidal selenium

[0119] (1) Weigh 1.2 g CTAB, 1 g PVP40 in a beaker, add 100 mL purified water, stir at room temperature for 10 min;

[0120] (2) Add 1.2 mL of 128 g / L concentration of selenious acid, stir and mix well at room temperature;

[0121] (3) Add 7 mL of 176 g / L concentration of ascorbic acid, stir and react at room temperature for 10 min to obtain colloidal selenium solution.

[0122] 3. Preparation of nitrocellulose membrane:

[0123] Dilute TP recombinant antigen 2 with coating solution to 0.8 mg / mL, and coat it on the detection line T1 position with a membrane marker at a parameter of 1 μL / cm;

[0124] Dilute HCV recombinant antigen 2 with coating solution to 1 mg / mL, and coat it on the detection line T2 position with a membrane marker at a parameter of 1 μL / cm;

[0125] Dilute goat anti-mouse IgG with coating solution to 1 mg / mL, and coat it on the control line position with a membrane marker at a parameter of 1 μL / cm; Place the coated nitrocellulose membrane in a 45°C oven and dry for 2 hours.

[0126] 4. Preparation method of colloidal selenium conjugate:

[0127] Colloidal selenium conjugate A preparation method: take 10 mL colloidal selenium solution in a beaker, after mixing 100 μL 0.2M potassium carbonate solution, add 300 μg SPA, 300 μg DNP-BSA, room temperature stirring reaction 10 min; add 0.5 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL stock solution, get colloidal selenium conjugate A;

[0128] Colloidal selenium conjugate B preparation method: take 10 mL colloidal selenium solution in a beaker, after mixing 100 μL 0.2M potassium carbonate solution, add 300 μg DNP, room temperature stirring reaction 10 min; add 0.5 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL stock solution, get colloidal selenium conjugate B; colloidal selenium conjugate A and colloidal selenium conjugate B according to the volume ratio 1:1.5 mixing, get colloidal selenium conjugate (i.e. colloidal selenium labeled SPA).

[0129] 5, colloidal selenium pad preparation method:

[0130] Colloidal selenium labeled conjugate (i.e. colloidal selenium labeled SPA) is diluted 40 times with stock solution, then soak the blank pad, take out and dry at 37℃ to get colloidal selenium pad.

[0131] 6, sample pad treatment: soak the blank pad in sample pad treatment solution, take out and dry at 37℃.

[0132] 7, test strip assembly: sample pad, colloidal selenium pad, nitrocellulose membrane and water absorption pad are sequentially pasted on the PVC bottom plate in the direction of chromatography, and cut into 3mm wide test strips.

[0133] Example 4: a kit for detecting syphilis spirochetes and hepatitis C virus in oral mucosa exudate based on colloidal selenium method

[0134] 1, solution preparation: 0.2M potassium carbonate configuration: 2.76g potassium carbonate in a beaker, add 100 mL purified water, stir and mix, then store at 4℃ for standby.

[0135] 10% BSA preparation: weigh 10g BSA in a beaker, add 100 mL purified water, stir and mix, then store at 4℃ for standby.

[0136] Coating solution preparation: weigh 2.4g Tris, 20g trehalose in a beaker, add 1L purified water, stir and mix, then adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0137] Preservation solution configuration: weigh 2.4g Tris, 8g BSA, 150g sucrose, 8g PEG20000 in a beaker, add purified water 1L, stir well, adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0138] Sample pad treatment solution configuration: weigh 20g Tris, 5g Casein, 5g Tween-20, 5g surfactant S7, 2.5g EDTA, add purified water 1L, stir well, adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0139] 2. Preparation of colloidal selenium

[0140] (1) Weigh 0.8g CTAB, 1.2g PVP40 in a beaker, add 100mL purified water, stir at room temperature for 10min;

[0141] (2) Add 0.8mL of 128g / L selenious acid, stir well at room temperature;

[0142] (3) Add 8mL of 176g / L ascorbic acid, stir at room temperature for 10min to obtain colloidal selenium solution.

[0143] 3. Preparation of nitrocellulose membrane:

[0144] Dilute TP recombinant antigen 2 to 1mg / mL with coating solution, use membrane marker to coat at 1μL / cm on the detection line T1 position;

[0145] Dilute HCV recombinant antigen 2 to 1mg / mL with coating solution, use membrane marker to coat at 1μL / cm on the detection line T2 position;

[0146] Dilute goat anti-mouse IgG to 1mg / mL with coating solution, use membrane marker to coat at 1μL / cm on the control line position; Place the coated nitrocellulose membrane in a 45℃ oven and dry for 2 hours.

[0147] 4. Preparation method of colloidal selenium conjugate:

[0148] Preparation method of colloidal selenium conjugate A: take 10mL colloidal selenium solution in a beaker, add 90μL 0.2M potassium carbonate solution, mix well, then add 280μg SPA, 280μg DNP-BSA, stir at room temperature for 10min; Add 1mL 10% BSA and block for 10min, centrifuge (temperature 4℃, speed 8500rpm, 15min) and discard the supernatant, add 1mL preservation solution to resuspend, to obtain colloidal selenium conjugate A;

[0149] Preparation method of colloidal selenium conjugate B: take 10 mL colloidal selenium solution in a beaker, after mixing 90 μL 0.2M potassium carbonate solution, add 280 μg DNP, stirring reaction at room temperature for 10 min; add 1 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL storage solution, get colloidal selenium conjugate B; colloidal selenium conjugate A and colloidal selenium conjugate B are mixed according to the volume ratio 1:2, get colloidal selenium conjugate (i.e. colloidal selenium labeled SPA).

[0150] 5. Preparation method of colloidal selenium pad:

[0151] The colloidal selenium labeled conjugate (i.e. colloidal selenium labeled SPA) is diluted 30 times with storage solution, then soak the blank pad, take out and dry at 37℃ to get colloidal selenium pad.

[0152] 6. Sample pad treatment: soak the blank pad in sample pad treatment solution, take out and dry at 37℃.

[0153] 7. Test strip assembly: sample pad, colloidal selenium pad, nitrocellulose membrane and water absorption pad are sequentially pasted on the PVC bottom plate in the direction of chromatography, cut into 3mm wide test strips.

[0154] Comparative example

[0155] Comparative example 1:

[0156] The difference between this comparative example and example 1 is that colloidal gold is used to label SPA.

[0157] Preparation method of colloidal gold conjugate A: take 10 mL colloidal gold solution in a beaker, after mixing 85 μL 0.2M potassium carbonate solution, add 250 μg SPA, 250 μg DNP-BSA, stirring reaction at room temperature for 10 min; add 0.5 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL storage solution, get colloidal gold conjugate A;

[0158] Preparation method of colloidal gold conjugate B: take 10 mL colloidal gold solution in a beaker, after mixing 85 μL 0.2M potassium carbonate solution, add 250 μg DNP, stirring reaction at room temperature for 10 min; add 0.5 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL storage solution, get colloidal gold conjugate B; colloidal gold conjugate A and colloidal gold conjugate B are mixed according to the volume ratio 1:1, get colloidal gold conjugate (i.e. colloidal gold labeled SPA).

[0159] Comparative example 2:

[0160] The difference between the present comparative example and Example 1 is that SDS and PEG20000 are used as templates to prepare the colloidal selenium solution, and the preparation method is as follows:

[0161] (1) 1 g of SDS and 1 g of PEG20000 were weighed into a beaker, 100 mL of purified water was added, and stirred at room temperature for 10 min;

[0162] (2) 1 mL of selenious acid with a mass concentration of 128 g / L was added, and stirred at room temperature;

[0163] (3) 5 mL of ascorbic acid with a mass concentration of 176 g / L was added, and stirred at room temperature for 10 min to obtain a colloidal selenium solution.

[0164] Comparative Example 3:

[0165] The difference between the present comparative example and Example 1 is that SDS and PVA are used as templates to prepare the colloidal selenium solution, and the preparation method is as follows:

[0166] (1) 1 g of SDS and 1 g of PVA were weighed into a beaker, 100 mL of purified water was added, and stirred at room temperature for 10 min;

[0167] (2) 1 mL of selenious acid with a mass concentration of 128 g / L was added, and stirred at room temperature;

[0168] (3) 5 mL of ascorbic acid with a mass concentration of 176 g / L was added, and stirred at room temperature for 10 min to obtain a colloidal selenium solution.

[0169] Comparative Example 4:

[0170] The difference between the present comparative example and Example 1 is that CTAB is used as a template to prepare the colloidal selenium solution, and the preparation method is as follows: (1) 1 g of CTAB was weighed into a beaker, 100 mL of purified water was added, and stirred at room temperature for 10 min;

[0171] (2) 1 mL of selenious acid with a mass concentration of 128 g / L was added, and stirred at room temperature;

[0172] (3) 5 mL of ascorbic acid with a mass concentration of 176 g / L was added, and stirred at room temperature for 10 min to obtain a colloidal selenium solution.

[0173] Comparative Example 5:

[0174] The difference between the present comparative example and Example 1 is that PVP40 is used as a template to prepare the colloidal selenium solution, and the preparation method is as follows:

[0175] (1) 1 g of PVP40 was weighed into a beaker, 100 mL of purified water was added, and stirred at room temperature for 10 min;

[0176] (2) Add 1 mL of selenious acid with a mass concentration of 128 g / L, stir and mix at room temperature;

[0177] (3) Add 5 mL of ascorbic acid with a mass concentration of 176 g / L, stir and react at room temperature for 10 min to obtain a colloidal selenium solution.

[0178] Comparative Example 6:

[0179] The difference between this comparative example and Example 1 is that CTAB and SDS are used as templates to prepare the colloidal selenium solution, and the preparation method is as follows:

[0180] (1) Take 1 g of CTAB and 1 g of SDS in a beaker, add 100 mL of purified water, stir and mix at room temperature for 10 min;

[0181] (2) Add 1 mL of selenious acid with a mass concentration of 128 g / L, stir and mix at room temperature;

[0182] (3) Add 5 mL of ascorbic acid with a mass concentration of 176 g / L, stir and react at room temperature for 10 min to obtain a colloidal selenium solution.

[0183] Comparative Example 7:

[0184] The difference between this comparative example and Example 1 is that PVP40 and SDS are used as templates to prepare the colloidal selenium solution, and the preparation method is as follows:

[0185] (1) Take 1 g of PVP40 and 1 g of SDS in a beaker, add 100 mL of purified water, stir and mix at room temperature for 10 min;

[0186] (2) Add 1 mL of selenious acid with a mass concentration of 128 g / L, stir and mix at room temperature;

[0187] (3) Add 5 mL of ascorbic acid with a mass concentration of 176 g / L, stir and react at room temperature for 10 min to obtain a colloidal selenium solution.

[0188] Comparative Example 8:

[0189] The difference between this comparative example and Example 2 is that DNP-BSA is not added when the colloidal selenium labels SPA, and DNP is not labeled separately. The specific preparation process is as follows:

[0190] 1. Solution preparation: 0.2M potassium carbonate preparation: 2.76g of potassium carbonate was added to a beaker with 100mL of purified water, stirred and mixed, and then stored at 4°C for standby.

[0191] 10% BSA preparation: 10g of BSA was weighed into a beaker, 100mL of purified water was added, stirred and mixed, and then stored at 4°C for standby.

[0192] Coating solution: weigh 2.4g Tris, 20g trehalose, in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0193] Preservation solution: weigh 2.4g Tris, 5g BSA, 100g sucrose, 5g PEG20000 in a beaker, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0194] Sample pad treatment solution: weigh 12g Tris, 5g Casein, 5g Tween-20, 5g surfactant S7, 2.5g EDTA, add purified water 1L, stir and mix well, then adjust pH to 8.0 with HCL solution, store at 4℃ for standby.

[0195] 2. Preparation of colloidal selenium

[0196] (1) Weigh 1g CTAB, 0.8g PVP40 in a beaker, add 100mL purified water, stir at room temperature for 10min;

[0197] (2) Add 1mL of selenious acid with a concentration of 128g / L, stir and mix well at room temperature;

[0198] (3) Add 10mL of ascorbic acid with a concentration of 176g / L, stir and react at room temperature for 10min to obtain colloidal selenium solution.

[0199] 3. Preparation of nitrocellulose membrane:

[0200] Dilute TP recombinant antigen 2 to 0.8mg / mL with coating solution, use membrane marker to coat at the position of detection line T1 with a parameter of 1μL / cm;

[0201] Dilute HCV recombinant antigen 2 to 0.8mg / mL with coating solution, use membrane marker to coat at the position of detection line T2 with a parameter of 1μL / cm;

[0202] Dilute goat anti-mouse IgG to 1mg / mL with coating solution, use membrane marker to coat at the position of quality control line with a parameter of 1μL / cm; Place the coated nitrocellulose membrane in an oven at 45℃ and dry for 2 hours.

[0203] 4. Preparation method of colloidal selenium conjugate:

[0204] Take 10 mL colloidal selenium solution in a beaker, add 70 μL 0.2M potassium carbonate solution mixed, then add 200 μg SPA, room temperature stirring reaction 10 min; add 0.5 mL 10% BSA blocking 10 min, centrifugation (temperature 4℃, speed 8500 rpm, 15 min) discard supernatant, add 1 mL stock solution, get colloidal selenium conjugate.

[0205] 5. Colloidal selenium pad preparation method:

[0206] The colloidal selenium labeled conjugate is diluted 20 times with the stock solution, then the blank pad is soaked, and the colloidal selenium pad is obtained by taking out and drying at 37℃.

[0207] 6. Sample pad treatment: soak the blank pad in the sample pad treatment solution, and take out and dry at 37℃.

[0208] 7. Test strip assembly: sequentially paste the sample pad, colloidal selenium pad, nitrocellulose membrane and water absorption pad on the PVC bottom plate in the chromatographic direction, and cut into test strips with a width of 3mm.

[0209] Performance test:

[0210] 1. Minimum detection limit test

[0211] Take TP positive samples for 10 times gradient dilution, get L1-L6, 6 gradient samples, take HCV positive samples for 10 times gradient dilution, get L7-L12, 6 gradient samples, detect 12 samples, the detection results are as follows:

[0212] Table 1 minimum detection limit test results

[0213]

[0214]

[0215] Note: "+" indicates positive, "-" indicates negative. From Table 1, the minimum detection limit of Example 1 is lower, and the sensitivity is 10 times higher than that of Comparative Example 1, indicating that the product prepared by the method of the application has higher sensitivity than the product prepared by colloidal gold.

[0216] The minimum detection limit of Example 1 is lower, and the sensitivity is 10 times higher than that of Comparative Example 2-7, indicating that the colloidal selenium particles prepared by the application using CTAB and PVP40 as templates are more uniform and stable, effectively improving the sensitivity of the product.

[0217] The sensitivity of Example 2 is 10 times higher than that of Comparative Example 8, which shows that, by adding DNP-BSA in the labeling process, and then mixing colloidal selenium conjugate A (SPA-colloidal selenium-DNP) with colloidal selenium conjugate B (colloidal selenium-DNP-BSA), and through the specific binding of DNP-BSA and DNP, the structure of colloidal selenium-DNP-BSA-DNP-colloidal selenium-SPA is formed, thereby increasing the number of colloidal selenium particles coupled to a single SPA protein, and when specific capture occurs at the detection line, more colloidal selenium particles are aggregated at the detection line, thereby enhancing positive color development and improving product sensitivity.

[0218] 2. Specificity and accuracy detection

[0219] The specificity detection method is as follows: 200 normal oral mucosa exudate samples are collected, and the kits produced by the examples and comparative examples are used for detection, respectively.

[0220] The accuracy detection method is as follows: 100 syphilis positive patient oral mucosa exudate samples and 100 hepatitis C positive patient oral mucosa exudate samples are collected, and the kits produced by the examples and comparative examples are used for detection, respectively.

[0221] Table 2: Detection results of the kit prepared in Example 1

[0222]

[0223] Table 3: Detection results of the kit prepared in Example 2

[0224]

[0225] Table 4: Detection results of the kit prepared in Example 3

[0226]

[0227] Table 5: Detection results of the kit prepared in Example 4

[0228]

[0229] Table 6: Detection results of the kit prepared in Comparative Example 1

[0230]

[0231] Table 7: Detection results of the kit prepared in Comparative Example 2

[0232]

[0233] Table 8: Detection results of the kit prepared in Comparative Example 3

[0234]

[0235] Table 9 detection results of the kit prepared in Comparative Example 4

[0236]

[0237] Table 10 detection results of the kit prepared in Comparative Example 5

[0238]

[0239] Table 11 detection results of the kit prepared in Comparative Example 6

[0240]

[0241] Table 12 detection results of the kit prepared in Comparative Example 7

[0242]

[0243] Table 13 detection results of the kit prepared in Comparative Example 8

[0244]

[0245] The specificity detection results are analyzed as follows:

[0246] As shown by the comparison between Table 2 and Table 6, the specificity of Example 1 is better than that of Comparative Example 1, indicating that the product prepared by the method of the application using colloidal selenium is more specific than the product prepared by colloidal gold.

[0247] As shown by the comparison between Table 2 and Table 7-12, the specificity of Example 1 is better than that of Comparative Examples 2-7, indicating that the colloidal selenium particles prepared by using CTAB and PVP40 as templates in the application are more uniform and stable, and effectively improve the specificity of the product.

[0248] As shown by the comparison between Table 3 and Table 13, the specificity of Example 2 is the same as that of Comparative Example 8, indicating that the addition of DNP-BSA and the separate labeling of DNP do not affect the specificity of the product when colloidal selenium is used to label SPA in the application.

[0249] The accuracy detection results are analyzed as follows:

[0250] As shown by Table 2-Table 5, the positive coincidence rate of Examples 1-4 is 100%, indicating that the syphilis spirochete and hepatitis C virus oral mucosa exudate antibody detection kits prepared by the colloidal selenium method in the application all meet the clinical needs.

[0251] The embodiments are only illustrative of the present application, and are not intended to limit the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A test strip for detecting Treponema pallidum, hepatitis C virus antibody in oral mucosal exudate based on colloidal selenium method, characterized in that: The test strip comprises a base plate, a sample pad, a colloidal selenium pad, a nitrocellulose membrane and a water absorption pad, the sample pad, the colloidal selenium pad, the nitrocellulose membrane and the water absorption pad are sequentially and adhesively connected on the base plate in a chromatographic direction, the nitrocellulose membrane is provided with a quality control line and two detection lines, the quality control line is coated with sheep anti-mouse IgG, and the two detection lines are respectively coated with TP recombinant antigen 2 and HCV recombinant antigen 2, and the colloidal selenium pad is coated with colloidal selenium-labeled SPA; ​ The preparation method of the colloidal selenium-labeled SPA is as follows: (1) potassium carbonate is added to the colloidal selenium solution and mixed, SPA and DNP-BSA are added and mixed, and then a blocking solution is added for reaction to obtain colloidal selenium conjugate A; (2) potassium carbonate is added to the colloidal selenium solution and mixed, DNP is added and mixed, and then a blocking solution is added for reaction to obtain colloidal selenium conjugate B; (3) colloidal selenium conjugate A and colloidal selenium conjugate B are mixed at a volume ratio of 1: (0.5-2) to obtain colloidal selenium-labeled SPA; In step (1), the addition amount of the colloidal selenium solution is 8-12 mL; the addition amount of potassium carbonate is 70-100 μL, the addition amount of SPA is 200-300 μg, the addition amount of DNP-BSA is 200-300 μg; and the addition amount of the blocking solution is 0.5-1 mL; In step (2), the addition amount of the colloidal selenium solution is 8-12 mL; the addition amount of potassium carbonate is 70-100 μL; the addition amount of DNP is 200-300 μg; and the addition amount of the blocking solution is 0.5-1 mL.

2. The test strip for detecting Treponema pallidum and hepatitis C virus antibody in oral mucous membrane exudate based on colloidal selenium method according to claim 1, characterized in that, In step (1), the mixing time of adding SPA and DNP-BSA is 10-20 min; and the reaction time of adding the blocking solution is 10-20 min.

3. The test strip for detecting Treponema pallidum and hepatitis C virus antibody in oral mucosal transudate based on colloidal selenium method according to claim 1, characterized in that, In step (2), the mixing time of adding DNP is 10-20 min; and the reaction time of adding the blocking solution is 10-20 min.

4. The test strip for detecting Treponema pallidum and hepatitis C virus antibody in oral mucosal transudate based on colloidal selenium method according to claim 1, characterized in that, The preparation process of the colloidal selenium solution is as follows: S1, cetyltrimethylammonium bromide and polyvinylpyrrolidone 40000 are mixed; S2, after mixing with selenous acid, ascorbic acid is added and mixed to obtain a colloidal selenium solution.

5. The test strip for detecting Treponema pallidum and hepatitis C virus antibody in oral mucosal transudate based on colloidal selenium method according to claim 4, characterized in that: In step S1, the addition amount of cetyltrimethylammonium bromide is 0.8-1.2 g; the addition amount of polyvinylpyrrolidone 40000 is 0.8-1.2 g; in step S2, the addition amount of selenous acid is 0.8-1.2 mL; and the addition amount of ascorbic acid is 5-10 mL.

6. The test strip for detecting Treponema pallidum and hepatitis C virus antibody in oral mucosal transudate based on colloidal selenium method according to claim 4, characterized in that, In step S1, the mixing time is 8-15 min; and in step S2, the mixing time of adding ascorbic acid is 8-15 min.

7. A kit characterized in that: The test strip of any one of claims 1-6. The test strip of any one of claims 1-6.

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