Chikungunya virus joint detection kit as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical detection, and particularly provides a chikungunya virus multi-target joint detection method based on a double-antibody sandwich immunochromatography technology and a rapid diagnosis kit. The synchronous high-sensitivity detection of virus infection is realized through a common detection line system for specifically recognizing the E1 protein and the C protein of the chikungunya virus. The kit has the characteristics that the performance index of the kit is close to the gold standard of nucleic acid detection, the kit is suitable for rapid screening of chikungunya virus infection, no special equipment is needed and the like, is particularly suitable for early screening and epidemiological monitoring of chikungunya virus infection in primary medical institutions and resource-limited areas, and provides reliable technical support for epidemic prevention and control.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunodetection, in particular to a co-detection kit for co-linear detection of chikungunya virus C protein and E1 protein by immunological technology, a preparation method and application thereof, which is suitable for rapid screening of chikungunya virus infection, especially for epidemic prevention and control in resource-limited areas. BACKGROUND

[0002] Chikungunya fever (CHIK) caused by chikungunya virus (CHIKV) infection is an acute infectious disease transmitted by Aedes mosquitoes, and its typical clinical features include fever, arthralgia and rash. In recent years, the epidemic range of the disease has continued to expand in the tropical and subtropical regions of the world. As a wide distribution area of Aedes aegypti and Aedes albopictus, China has experienced local transmission of imported cases several times.

[0003] Currently, the clinical diagnosis of chikungunya virus infection mainly relies on three types of detection methods: nucleic acid detection, which uses real-time fluorescent PCR technology, although it has high specificity, but it requires professional equipment and operators, and it is difficult for primary medical institutions to popularize; virus isolation, which relies on cell culture, takes 3-7 days and the sensitivity is significantly affected by the quality of the sample; serological detection, ELISA method is complex to operate, mainly based on E1 protein, has a diagnostic window period, and has a high rate of missed detection. And the genetic variation of virus E1 protein (such as E1-A226V mutation of the Indian Ocean branch) can change the antigenic epitope, further reducing the detection accuracy.

[0004] In view of the above problems, the development of rapid and high-sensitivity on-site detection tools has become the key to prevention and control. The immunochromatographic test strip technology is listed as the preferred scheme due to its simple operation and low cost. However, the detection based on E1 protein has a high rate of missed detection. E1 protein is highly expressed in the early stage of infection (1-7 days), and the capsid protein C protein can still be detected in the middle and late stages of infection (after 5 days), and the complement of the two can cover the entire diagnostic window period. If C protein is combined, the detection performance can be improved.

[0005] The co-detection product designed with multiple detection lines has two major defects: one is a high error rate in interpretation by non-professionals, and the other is an increase in production cost of 30% due to the increase in the area of the test strip. Therefore, the development of a new detection system that can simultaneously capture E1 / C protein antigens on a single detection line has become a key path to break through the bottleneck of existing technology. SUMMARY

[0006] The purpose of the present application is to provide a co-detection kit for virus detection, in particular a co-detection kit for chikungunya virus with high sensitivity and specificity.

[0007] Another purpose of the present application is to provide a preparation method of the co-detection kit.

[0008] Another object of the present application is to provide an application of the combined detection kit.

[0009] The technical scheme adopted to achieve the object of the present application is as follows:

[0010] A combined detection kit for detection, comprising a reagent strip, wherein the reagent strip comprises:

[0011] a binding pad coated with a first protein labeled with a marker, a second protein labeled with a marker, a biotinylated first protein and a biotinylated second protein;

[0012] a detection pad, which is a nitrocellulose membrane provided with a single detection line, wherein the detection line is provided with a capture molecule capable of capturing biotin;

[0013] a sample pad and an absorbent pad.

[0014] In the above technical scheme, the reagent strip comprises a sample pad, a binding pad, a detection pad and an absorbent pad connected in sequence.

[0015] The reagent strip is provided with only one detection line, and the detection line is fixed with a capture molecule capable of binding with the first protein and the second protein.

[0016] The reagent strip can simultaneously detect substances capable of binding with the first protein and the second protein.

[0017] The combined detection kit is for Chikungunya virus, the first protein is an E1 protein antibody, and the second protein is a C protein antibody.

[0018] The marker is selected from colloidal gold, latex microspheres or fluorescent microspheres;

[0019] The capture molecule is selected from streptavidin or an anti-biotin antibody.

[0020] The reagent strip is further provided with a quality control line coated with a coating protein selected from Protein G, goat anti-mouse IgG and streptococcal protein G.

[0021] The kit further comprises a sample processing reagent.

[0022] The sample is subjected to lysis treatment.

[0023] The sample processing reagent comprises Tris, NaCl, Triton X-100 and BSA.

[0024] Another aspect of the present application provides a preparation method of the combined detection kit, comprising the following steps:

[0025] Step 1: coating the detection line: a substance capable of capturing biotin is prepared into a coating solution to coat the detection line of the nitrocellulose membrane.

[0026] Step 2, binding pad processing: E1 protein antibody labeled with a marker, C protein antibody labeled with a marker, biotinylated E1 protein antibody and biotinylated C protein antibody are coated on the binding pad.

[0027] Step 3, quality control line coating: Protein G is prepared into a coating solution and coated on the quality control line of the nitrocellulose membrane.

[0028] The amount of Protein G coated on the quality control line is 2 mg / mL.

[0029] In another aspect of the present application, a detection method for non-diagnostic purposes based on the above-mentioned combined detection kit is provided, comprising the following steps:

[0030] Step S1, mixing the sample to be tested with a sample processing solution;

[0031] Step S2, adding the mixed solution to the sample pad of the kit;

[0032] Step S3, observing the color development of the detection line and the quality control line after 15 minutes of reaction.

[0033] In still another aspect of the present application, the application of the above-mentioned combined detection kit in the preparation of a Chikungunya virus detection product is provided.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] 1. A new co-line combined detection scheme is established, and through the optimized co-line label design, the anti-first protein and the anti-second protein monoclonal antibodies are captured on the same detection line (T line) to form a "double antigen capture complex", and only one color development line is needed to indicate the presence of the first protein and the second protein. The co-line combined detection result is simple to read, and it is convenient for non-professionals to test themselves;

[0036] 2. In the detection of Chikungunya virus, E1 protein and C protein are co-detected, which covers the detection requirement of E1 deletion strain and complements the complete diagnostic window period, with low missed detection rate;

[0037] 3. The area of the reagent strip required for co-line detection is small, which reduces the production cost.

[0038] 5. The detection time of the combined detection kit of the present application is only 15 minutes, and compared with the C protein single detection reagent strip, the risk of missing Chikungunya virus detection is reduced by 47%, which improves the accuracy and efficiency of Chikungunya virus detection and diagnosis.

[0039] In conclusion, the application provides a new virus co-detection kit, and also provides a new co-detection kit for CHIKV, realizes co-linear co-detection of E1 protein and C protein, and provides a simple, rapid, sensitive and specific scheme for detection of CHIKV. DETAILED DESCRIPTION

[0040] Hereinafter, the application will be described in detail. It should be understood that the following description is only illustrative of the application and is not intended to limit the scope of the application, which is defined by the appended claims. Moreover, those skilled in the art will appreciate that modifications can be made to the application without departing from the spirit and scope thereof. If not specifically mentioned, the technical means used in the examples are conventional means known to those skilled in the art.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Prior to the detailed description of the application, the following definitions are provided to better understand the application.

[0042] In the event that a range of values is provided, for example, a concentration range, a percentage range or a ratio range, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, and any other stated or intervening value or sub-range therein are encompassed within the subject matter. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges, and are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.

[0043] In the context of the present application, many embodiments use the expressions "comprising", "including" or "consisting essentially of". The expressions "comprising", "including" or "consisting essentially of" can be understood in the general case as open-ended expressions, meaning that not only the elements, components, assemblies, method steps etc. specifically listed after the expression are included, but also other elements, components, assemblies, method steps. In addition, in the present document, the expressions "comprising", "including" or "consisting essentially of" can also be understood in some cases as closed expressions, meaning that only the elements, components, assemblies, method steps specifically listed after the expression are included, and not any other elements, components, assemblies, method steps. In this case, the expression is equivalent to the expression "consisting of".

[0044] For better understanding of the present teachings and without limiting the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or ratios and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of significant digits it contains and by applying ordinary rounding techniques.

[0045] E1 protein (envelope glycoprotein E1) is the main component of the viral envelope, expressed in large quantities in the early stage of infection, easily recognized by the host immune system, and induces strong antibody response (IgM / IgG). Suitable for acute phase detection: viremic phase (1-7 days after onset). E1 causes high antibody titer and can be used as a target for IgG and IgM detection in later antibody detection.

[0046] C protein (capsid protein) antigen content is high, suitable for rapid antigen detection, still detectable after the late stage of viral replication (5 days later), and can be used as a core target for antigen detection.

[0047] Due to the point mutation of E1 protein in some epidemic strains (such as the Indian Ocean branch), which may affect antibody binding and cause escape. The detection of C protein can cover sensitivity and variant strain coverage, which is complementary to E1 protein and can significantly improve the reliability of detection. The double detection line design needs to distinguish T1 (E1 protein) and T2 (C protein) bands, which is complex to interpret and easy to misjudge for non-professionals. In addition, double-line detection requires a larger reagent strip area, increasing production costs.

[0048] In summary, the present application aims to provide a virus co-detection kit that is easy to interpret. The present application also provides a co-detection kit for Chikungunya virus based on this scheme.

[0049] Therefore, in a first aspect, the present application provides a co-detection kit for viruses, comprising a reagent strip, the reagent strip comprising:

[0050] a conjugate pad coated with a first protein labeled with a marker, a second protein labeled with a marker, a biotinylated first protein and a biotinylated second protein;

[0051] a nitrocellulose membrane provided with a single detection line, the detection line being coated with a substance capable of capturing biotin;

[0052] a sample pad and a water absorption pad.

[0053] The reagent strip is provided with only one detection line, and the reagent strip can simultaneously detect substances that can bind to the first protein and the second protein.

[0054] In one embodiment, the first protein is an antibody and the second protein is an antibody.

[0055] In one embodiment, the first protein is an antigen and the second protein is an antibody.

[0056] In one embodiment, the virus is CHIKV, the first protein is an E1 protein antibody and the second protein is a C protein antibody.

[0057] In one embodiment, the virus is CHIKV, the first protein is an E2 protein antibody and the second protein is a C protein antibody.

[0058] In one embodiment, the virus is CHIKV, the first protein is an E1 antigen and the second protein is a C protein antibody.

[0059] The reagent strip can either detect antigen with antibody or detect antibody with antigen.

[0060] In one embodiment, the E1 antibody is an anti-E1 protein monoclonal antibody.

[0061] In one embodiment, the biotinylated antibody is directly linked by chemical cross-linking between biotin and antibody.

[0062] In one embodiment, the reagent strip comprises, in order, a sample pad, a conjugate pad, a detection pad (nitrocellulose membrane) and an absorbent pad.

[0063] The detection pad comprises a test line (T line) and a control line (C line).

[0064] In one embodiment, the label is colloidal gold.

[0065] In another embodiment, the label is a latex microsphere. The label can also be a fluorescent microsphere or other particle that can serve as an indicator.

[0066] In another embodiment, the label is a fluorescent microsphere.

[0067] In one embodiment, the biotin-capturable substance is streptavidin.

[0068] In another embodiment, the biotin-capturable substance is an anti-biotin antibody, avidin, or other substance that can capture biotin.

[0069] The coating protein of the reagent strip quality control line (C line) can be selected according to the antibody labeling type of the detection system. In a specific embodiment, the coating protein of the quality control line is Protein G.

[0070] The coating protein of the quality control line can also be goat anti-mouse IgG antibody or streptococcal protein G.

[0071] The kit also includes a sample processing solution.

[0072] The sample is subjected to lysis treatment.

[0073] In a specific embodiment, the sample processing reagent includes Tris, NaCl, Triton X-100, and BSA.

[0074] In a specific embodiment, the sample processing method is as follows: the sample to be tested is added to a certain amount of sample processing solution and mixed thoroughly.

[0075] In a second aspect, the present application provides a preparation method of the combined detection kit, comprising the following steps:

[0076] Step 1, detection line coating: a biotin-capturable substance is prepared into a coating solution, and the detection line of the nitrocellulose membrane is coated.

[0077] Step 2, binding pad treatment: the marker-labeled E1 protein antibody, the marker-labeled C protein antibody, the biotinylated E1 protein antibody, and the biotinylated C protein antibody are coated on the binding pad.

[0078] Step 3, quality control line coating: Protein G is prepared into a coating solution, and the quality control line of the nitrocellulose membrane is coated.

[0079] The coating concentration of Protein G on the quality control line is 1.5-2.5 mg / mL.

[0080] In a specific embodiment, the coating concentration of Protein G on the quality control line is 2 mg / mL.

[0081] In a third aspect, the present application provides a non-diagnostic purpose detection method based on the combined detection kit, comprising the following steps:

[0082] Step S1, mixing the sample to be tested with the sample processing solution;

[0083] Step S2, adding the mixed solution dropwise to the sample pad of the kit;

[0084] Step S3, observing the color development of the detection line and the quality control line after 15 minutes of reaction.

[0085] The fourth aspect of the present application provides a use of the combined detection kit in the preparation of a Chikungunya virus detection product.

[0086] The combined detection kit of the present application is developed by using the principle of immunochromatography technology and sandwich immunoassay. The detection line on the nitrocellulose membrane of the test paper is coated with streptavidin which can capture biotinylated antibody, and the quality control line is coated with protein such as Protein G which can bind antibody. The specific detection antibody labeled with colloidal gold and the biotinylated specific detection antibody are pre-coated on the conjugate pad. During detection, the sample to be tested is added to the sample pad, and then climbs along the reagent strip to contact the antibody-colloidal gold complex and the antibody-biotin complex on the detection pad. If the sample contains E1 protein and / or C protein, the E1 protein and / or C protein binds with the antibody-colloidal gold complex and the biotin antibody complex to form an Ab-Ag-Ab sandwich complex. The sandwich complex continues to climb to the detection line on the nitrocellulose membrane and is collected with the streptavidin pre-coated on the detection line. The Ab-Ag-Ab-colloidal gold complex accumulates and develops color to form a distinct micro-red band, which is the T line, representing a positive result. If the sample does not contain E1 protein and / or C protein, no distinct red band will be formed at the detection line position, which represents a negative result. The free complex continues to climb to the quality control line on the test paper and is collected with the pre-coated protein such as Protein G to form a micro-red band, which is the C line, representing the quality control line.

[0087] The present application will be further described in conjunction with specific examples. It should be understood that the specific examples described herein are intended to serve only to explain the present application and are not intended to limit the present application.

[0088] Examples

[0089] In the following examples, the preparation method of the antibody of the present application and the characterization of the related properties are shown. Unless otherwise specified, the experimental methods used herein are conventional methods, and unless otherwise specified, the experimental materials used in the following examples are obtained from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0090] It should be noted that the terms used in the specification of the present application are only for the purpose of describing the specific embodiments and are not intended to limit the present application. The summary, detailed description and examples above are only for the purpose of specifically illustrating the present application and are not intended to limit the present application in any way. The scope of the present application is determined by the appended claims without departing from the spirit and essential characteristics of the present application.

[0091] Example 1

[0092] A kit for simultaneous detection of Chikungunya virus, the reagent strip of which comprises, in order, a sample pad, a conjugate pad, a detection pad and an absorbent pad.

[0093] The conjugate pad is coated with E1 protein antibody labeled with colloidal gold, C protein antibody labeled with colloidal gold, biotinylated E1 protein antibody and biotinylated C protein antibody;

[0094] The detection pad (nitrocellulose membrane) is provided with a quality control line and a single detection line, the quality control line is coated with 2 mg / mL Protein G, and the detection line is coated with 2 mg / mL streptavidin (heavy chain biological, HP155-3);

[0095] The kit also includes a sample processing reagent, the sample processing solution contains Tris, NaCI, Triton X-100 and BSA, the sample to be tested is mixed with the sample processing solution, and then the mixture is added dropwise to the sample pad of the kit; after 15 minutes of reaction, the color development of the detection line and the quality control line is observed to determine the result.

[0096] The conjugate pad and the detection pad of the kit are provided as shown in Table 1.

[0097] Table 1

[0098]

[0099] Example 2

[0100] Assembly of colloidal gold-based simultaneous detection kit

[0101] Preparation of colloidal gold: add 100 mL of ultrapure water to a triangular flask, heat to boiling, add 2 mL of 2% chloroauric acid solution (Sigma-Aldrich, Catalog No.: 16961-25-4), immediately after boiling, add 1 mL of 2% trisodium citrate (Sigma-Aldrich, Catalog No.: 6132-04-3) aqueous solution, continue to stir and boil for 10 minutes, and cool naturally for standby.

[0102] Antibody colloidal gold complex: take 10 mL of the above colloidal gold into a beaker, adjust the pH to 7.0 by adding 120 μL of 0.2M K2CO3 under stirring, continue to stir for 10 seconds; add 100 μg of E1 antibody, continue to stir for 5 minutes; add 0.1 mL of 10% BSA, continue to stir for 5 minutes; centrifuge at 12000g for 10 minutes, discard the supernatant, and dilute the precipitate to 1 mL with colloidal gold diluent (10 mM PB, 150 mM NaCl, 0.2% BSA, 0.1% Triton X-100, 3% Sucrose, 0.01% Proclin 300) as anti-E1 protein antibody colloidal gold complex. In the same way, prepare the anti-C protein antibody colloidal gold complex.

[0103] Antibody biotin complex: take 100 ug of E1 antibody, add 20 μL of 5 mM biotin, react at room temperature for 1 hour in the dark, add 1 M Tris-HCl (pH 7.5) to a final concentration of 50 mM, terminate the reaction; remove free biotin with a desalting column, collect the E1 protein antibody biotin complex, and reserve for use. In the same way, prepare the anti-C protein antibody biotin complex.

[0104] Mix the two antibody colloidal gold complexes and the two antibody biotin complexes in equal amounts, spray the pads at a final concentration of 2 mg / mL, and after drying, prepare the binding pad.

[0105] Detection pad: dilute SA (HP155-3) 2 mg / mL to prepare the working solution for the detection line, draw a line on the nitrocellulose membrane using a membrane marker, and dry at 50°C for 1 hour to form the detection line (T line); dilute Protein G (DC166) 2 mg / mL to prepare the working solution for the quality control line, draw a line on the nitrocellulose membrane using a membrane marker, and dry at 50°C for 1 hour to form the quality control line (C line).

[0106] Paste the sample pad, the binding pad, the detection pad, and the water absorption pad on the base plate in that order, cut them into 4 mm strips, and put them into the box to form the test kit.

[0107] Example 3: E1 protein immunochromatographic kit based on colloidal gold

[0108] Prepare the colloidal gold according to the method described in Example 2.

[0109] Antibody colloidal gold complex: take 10 mL of the above colloidal gold into a beaker, adjust the pH to 7.0 by adding 120 μL of 0.2 M K2CO3 while stirring, continue stirring for 10 seconds; add 100 μg of E1 antibody, continue stirring for 5 minutes; add 0.1 mL of 10% BSA, continue stirring for 5 minutes; centrifuge at 12000 g for 10 minutes, discard the supernatant, and dilute the precipitate to 1 mL with colloidal gold diluent (10 mM PB, 150 mM NaCl, 0.2% BSA, 0.1% Triton X-100, 3% sucrose, 0.01% Proclin 300) to obtain the anti-E1 protein antibody colloidal gold complex.

[0110] Antibody biotin complex: take 100 ug of E1 antibody, add 20 μL of 5 mM biotin, react at room temperature for 1 hour in the dark, add 1 M Tris-HCl (pH 7.5) to a final concentration of 50 mM, terminate the reaction; remove free biotin with a desalting column, collect the E1 protein antibody biotin complex, and reserve for use.

[0111] Mix the E1 protein antibody colloidal gold complex and the E1 protein antibody biotin complex in equal amounts, spray the pad at a final concentration of 2 mg / mL, and dry to prepare the conjugate pad.

[0112] Detection pad: complete according to the method described in Example 2;

[0113] Paste the sample pad, the conjugate pad, the detection pad, and the water absorption pad on the base plate in order, cut them into 4 mm reagent strips, and load them into the box body to become a detection kit.

[0114] Example 4 C protein immunochromatographic kit based on fluorescent microspheres

[0115] Fluorescent microsphere activation: take 1 mg of fluorescent microspheres (about 1 mL of suspension), centrifuge (12,000 rpm, 10 minutes) and discard the supernatant; wash twice with 50 mM MES (pH 6.0) and resuspend in 200 μL of MES; add 10 μL of EDC (10 mg / mL) and 10 μL of NHS (10 mg / mL), and shake at room temperature for 30 minutes.

[0116] Antibody fluorescent microsphere coupling: mix the activated microspheres with the antibody at a mass ratio of 1:5 in PBS (pH 7.4), 100 ug of C protein antibody, add 20 μg of microspheres, react at 4°C for 2 hours, and shake gently; add 10 μL of 1M glycine (to terminate the reaction), incubate for 30 minutes, centrifuge and wash 3 times (PBS + 0.1% BSA), remove the free antibody, resuspend in PBS containing 1% BSA and 0.05% NaN3, and store at 4°C in the dark.

[0117] Antibody biotin complex: take 100 ug of C protein antibody, add 20 μL of 5 mM biotin, react at room temperature for 1 hour, add 1M Tris-HCl (pH 7.5) to a final concentration of 50 mM, and terminate the reaction; remove the free biotin with a desalting column, collect the C protein antibody biotin complex, and reserve.

[0118] Mix the C protein antibody fluorescent microsphere complex and the C protein antibody biotin complex in equal amounts, spray the pad at a final concentration of 2 mg / mL, and dry to prepare the conjugate pad.

[0119] Detection pad: complete according to the method described in Example 2;

[0120] Paste the sample pad, the conjugate pad, the detection pad, and the water absorption pad on the base plate in order, cut them into 4 mm reagent strips, and load them into the box body to become a detection kit.

[0121] Example 5

[0122] The method for using the combined detection kit includes the following steps:

[0123] 1. Collect and process samples: add patient serum to sample processing solution, mix well;

[0124] 2. Add the processed samples to the sample pad sample wells of the co-detection kit of Example 2, respectively, and wait for 15 minutes;

[0125] 3. Observe and record the results, and the test line T line color development is positive for Chikungunya virus.

[0126] Example 6

[0127] Using the detection scheme of Example 5, 40 positive serum samples and 200 negative serum samples collected clinically were detected, and the detection results of the serum samples by nucleic acid detection (gold standard), the kits of Example 2, Example 3 and Example 4 are shown in Table 2, Table 3, Table 4 and Table 5. The nucleic acid test uses Shengling Medical Chikungunya Virus Nucleic Acid Detection Kit for detection, and the operation is according to its instruction manual. The standard for positive determination is that the detection result Ct value is ≤37, and there is obvious exponential growth.

[0128] Table 2 Nucleic acid detection results

[0129]

[0130] The sensitivity of nucleic acid detection is 95%, the specificity is 99.0%, and the total coincidence rate is 98.3%.

[0131] Table 3 Detection results of Example 2 co-detection kit

[0132]

[0133] The sensitivity is 91%, the specificity is 98%, and the coincidence rate is 96.7%.

[0134] Table 4 Detection results of Example 3 kit

[0135]

[0136] The sensitivity is 63%, the specificity is 96%, and the total coincidence rate is 90.4%.

[0137] Table 5 Detection results of Example 4 kit

[0138]

[0139] The sensitivity is 83%, the specificity is 95.0%, and the total coincidence rate is 92.9%.

[0140] Table 6 Result analysis

[0141]

[0142] As above, the kit performance of Example 2 is close to nucleic acid detection, without relying on professional equipment, and the detection result can be obtained within 15 minutes through simple operation, and the result interpretation is intuitive and clear, and non-professionals can also accurately operate; while maintaining high specificity (98%), the sensitivity reaches 91%, the total coincidence rate of detection is 96.7%, and the performance is close to the gold standard of nucleic acid detection (98.3%).

[0143] The co-line joint detection kit of Example 2 has obvious cost advantage compared with the scheme of using multiple reagent strips to detect multiple indicators. The integrated design integrates multiple detection indicators on a single detection line, and the detection line is colored to be positive. It is especially suitable for non-professionals to use.

[0144] The colloidal gold chromatography detection of Example 3 for E1 protein has low sensitivity and still has room for optimization.

[0145] The separate detection scheme of Example 4 for C protein also has certain significance and can be used as a preliminary screening scheme.

[0146] Example 7

[0147] The kit of Example 2 was tested in parallel using the antigens shown in Table 7 (5 ng / mL recombinant protein) as samples to detect whether there was cross reaction.

[0148] Table 7

[0149]

[0150] The results show that the above items have no cross reaction.

[0151] The above content is only an exemplary description of the present application. Based on the understanding of the core idea of the present application, those skilled in the art can appropriately adjust and optimize the specific embodiments. Any reasonable modification, equivalent replacement or technical extension based on the technical scheme of the present application shall be included in the protection scope of the present application.

Claims

1. A combined reagent kit for virus detection, characterized by, The kit comprises a reagent strip, which comprises: a conjugate pad coated with a first protein labeled with a marker, a second protein labeled with a marker, a biotinylated first protein and a biotinylated second protein; a nitrocellulose membrane provided with a single detection line coated with a capture molecule capable of capturing biotin; a sample pad and a water absorption pad.

2. The combined test kit according to claim 1, characterized by The virus is Chikungunya virus, the first protein is an E1 protein antibody, and the second protein is a C protein antibody.

3. The combined test kit according to claim 1 or 2, characterized in that, The marker is selected from colloidal gold, latex microspheres or fluorescent microspheres.

4. The combined test kit according to claim 1 or 2, characterized in that, The capture molecule is selected from streptavidin or an anti-biotin antibody.

5. The combined test kit according to claim 1 or 2, characterized in that, The reagent strip is further provided with a quality control line coated with a coating protein.

6. The kit according to claim 5, wherein the coating protein is selected from Protein G, goat anti-mouse IgG or streptococcal protein G.

7. The combined test kit according to claim 1 or 2, characterized in that, The kit further comprises a sample processing solution.

8. A method for preparing the combined test kit according to any one of claims 1 to 7, characterized in that, The kit comprises the following steps: Step 1, detection line coating: prepare a coating solution of a biotin-capturing substance, and coat the nitrocellulose membrane by line marking; Step 2, conjugate pad treatment: coat the marker-labeled E1 protein antibody, the marker-labeled C protein antibody, the biotinylated E1 protein antibody and the biotinylated C protein antibody on the conjugate pad. Step 3, quality control line coating: prepare a coating solution of Protein G, and coat the nitrocellulose membrane by line marking.

9. A method for non-diagnostic purposes based on the combined test kit according to any one of claims 1 to 7, characterized in that, The kit comprises the following steps: Step S1, mix the sample to be tested with the sample processing solution; Step S2, drop the mixed solution onto the sample pad of the kit; Step S3, observe the color development of the detection line and the quality control line after 15 minutes of reaction.

10. Use of the kit according to any one of claims 1-7 in the preparation of a Chikungunya virus detection product.

Images