Chikungunya virus antigen and dengue virus antigen detection kit, preparation method and detection method

Abstract

The invention relates to a Chikungunya virus antigen and dengue virus antigen detection kit, a preparation method and a detection method. The kit comprises a chikungunya virus antibody marked by quantum dot microspheres, a dengue virus antibody marked by quantum dot microspheres, a sample loading solution and immunochromatography detection test paper. The kit and the detection method based on the fluorescence immunochromatography are high in sensitivity, strong in specificity and good in stability, can realize synchronous quantitative detection of chikungunya virus antigen and dengue virus antigen, effectively improve the detection efficiency, are simple and efficient in detection operation process, can report a result within 15 minutes, are suitable for on-site rapid detection, and have good application prospects. And a new means is provided for prevention, control, diagnosis and treatment of chikungunya virus and dengue virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a chikungunya virus antigen and dengue virus antigen detection kit, preparation method and detection method. Background Technology

[0002] Chikungunya virus: Belongs to the family Phaeoviridae, genus Alphavirus. Its typical characteristic is causing severe joint pain and arthritis lasting for weeks or even years, with a high rate of disability, but rarely fatal. Dengue virus: Belongs to the family Flaviviridae, genus Flavivir. Its greatest risk lies in the potential development of dengue hemorrhagic fever or dengue shock syndrome, which is life-threatening and requires close monitoring. Because both viruses are transmitted by Aedes mosquitoes (mainly Aedes aegypti and Aedes albopictus), their geographical distribution and epidemic seasons highly overlap, and their acute symptoms (such as high fever, headache, rash, and muscle and joint pain) are very similar, their disease management and prognosis are drastically different. Furthermore, co-infection with these two viruses occurs frequently; therefore, rapid and accurate differential diagnosis is crucial.

[0003] Currently, the main detection methods for chikungunya virus and dengue virus include molecular detection, serological detection, and antigen detection. Molecular detection directly detects the virus's genetic material, specifically including real-time quantitative RT-PCR, multiplex RT-PCR, and isothermal amplification techniques. For example, KR102468964B1 discloses a primer set for diagnosing various mosquito-borne infectious diseases, including dengue fever, Zika, and chikungunya, and a simultaneous multi-molecular diagnostic method using it. However, molecular detection methods typically require specialized personnel and expensive equipment, and the testing time is long, making them unsuitable for rapid on-site testing. Serological detection detects antibodies produced after the body's immune response, such as IgM / IgG antibody detection; however, antibody production takes time, with a "window period." Antigen detection, on the other hand, has the advantages of being rapid, sensitive, and highly specific, and can assist in early diagnosis of viral infection, helping to facilitate timely treatment and control measures, making it suitable for rapid on-site testing.

[0004] Immunochromatography is a widely used rapid on-site detection method; however, traditional immunochromatography uses colloidal gold as an immunomarker, which has low sensitivity and cannot provide quantitative detection. Therefore, developing technologies capable of rapidly and accurately quantifying chikungunya virus antigen and dengue virus antigen is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a chikungunya virus antigen and dengue virus antigen detection kit, preparation method, and detection method. It develops a technical solution for detecting chikungunya virus antigen and dengue virus antigen based on quantum dot fluorescence immunochromatography, aiming to achieve rapid, accurate, and low-cost detection of chikungunya virus antigen and dengue virus antigen.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a detection kit for chikungunya virus antigen and dengue virus antigen, the kit comprising quantum dot microsphere-labeled chikungunya virus antibody, quantum dot microsphere-labeled dengue virus antibody, sample loading solution, and immunochromatographic test strip; the immunochromatographic test strip is composed of chikungunya virus antigen test strip and dengue virus antigen test strip arranged side by side; the detection line of the chikungunya virus antigen test strip is coated with chikungunya virus capture antibody, and the control line is coated with goat anti-mouse IgG polyclonal antibody; the detection line of the dengue virus antigen test strip is coated with dengue virus capture antibody, and the control line is coated with goat anti-mouse IgG polyclonal antibody.

[0008] This invention designs a reagent kit based on fluorescence immunochromatography, which has high sensitivity, strong specificity, and good stability. It can realize the simultaneous quantitative detection of chikungunya virus antigen and dengue virus antigen, effectively improving the detection efficiency. Moreover, the detection operation process is simple and efficient, and the results can be reported within 15 minutes, making it suitable for rapid on-site detection.

[0009] Optionally, the loading solution contains Tris-HCl, sucrose, trehalose, polyethylene glycol, bovine serum albumin, and Tween.

[0010] Optionally, the polyethylene glycol includes PEG-2000.

[0011] Optionally, the Tween includes Tween-20.

[0012] Optionally, the loading solution contains 0.025~0.15 mol / L Tris-HCl (e.g., 0.03, 0.04, 0.05, 0.08, 0.1, 0.12, or 0.14 mol / L), 0.5~6 wt% sucrose (e.g., 0.8 wt%, 1 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, or 5.5 wt%), 0.5~6 wt% trehalose (e.g., 0.8 wt%, 1 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, or 5.5 wt%), and 0.25~1 wt% polyethylene glycol (e.g., 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%). The following are the components: 0.1-1 wt% bovine serum albumin (e.g., 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, or 0.9 wt%) and 0.5-3 wt% Tween (e.g., 0.6 wt%, 0.7 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 2.8 wt%, or 2.9 wt%); preferably 0.05 mol / L Tris-HCl, containing 3% sucrose, 3% trehalose, 0.5% PEG-2000, 0.5% BSA, and 1% Tween-20.

[0013] Optionally, the pH of the loading solution is 5.5 to 8.0, preferably pH 6.5.

[0014] In this invention, the influencing factors of fluorescence signal during the detection process are analyzed in depth, and a suitable sample loading solution is designed to further improve the detection fluorescence signal response value.

[0015] Optionally, the chikungunya virus antigen test strip and the dengue virus antigen test strip each independently include a base plate, a sample pad, a detection membrane, and absorbent paper, wherein the sample pad, the detection membrane, and the absorbent paper are sequentially connected and arranged on the base plate.

[0016] Optionally, the detection membrane comprises a nitrocellulose membrane.

[0017] Optionally, the nitrocellulose membrane includes any one of CN140 membrane, CN95 membrane, or Millipore 135 membrane, preferably CN95 membrane.

[0018] Optionally, the detection line of the chikungunya virus antigen test strip is coated with chikungunya virus capture antibody at a concentration of 0.5~1.5 mg / mL (e.g., 0.6, 0.8, 1, 1.2 or 1.4 mg / mL, etc.), preferably 0.8 mg / mL.

[0019] Optionally, the dengue virus antigen test strip has a dengue virus capture antibody concentration of 0.5 to 1.5 mg / mL (e.g., 0.6, 0.8, 1, 1.2 or 1.4 mg / mL, etc.), preferably 0.8 mg / mL.

[0020] In this invention, a specific antibody coating concentration for the detection line is designed to further improve the detection fluorescence signal response value.

[0021] Optionally, the coating is performed using a film-spreading machine at a speed of 0.5~2 µL / cm, preferably 1 µL / cm.

[0022] Optionally, the quantum dot microsphere-labeled chikungunya virus antibody binds to the chikungunya virus at a different site than the chikungunya virus capture antibody, and the quantum dot microsphere-labeled dengue virus antibody binds to the dengue virus at a different site than the dengue virus capture antibody.

[0023] In a second aspect, the present invention provides a method for preparing a detection kit for chikungunya virus antigen and dengue virus antigen as described in the first aspect, the method comprising: combining the quantum dot microsphere-labeled chikungunya virus antibody, the quantum dot microsphere-labeled dengue virus antibody, the sample loading solution and the immunochromatographic test strip.

[0024] Thirdly, the present invention provides a method for detecting chikungunya virus antigen and dengue virus antigen for non-disease diagnosis and / or treatment purposes. The detection method utilizes the detection kit for chikungunya virus antigen and dengue virus antigen described in the first aspect, and includes:

[0025] The quantum dot microsphere-labeled Chikungunya virus antibody and the quantum dot microsphere-labeled dengue virus antibody were mixed with the sample loading solution to obtain Chikungunya virus loading solution and dengue virus loading solution, respectively. These were then mixed with the samples to be tested and added to the Chikungunya virus antigen test strip and the dengue virus antigen test strip, respectively. The fluorescence of the test line and the control line was observed.

[0026] This invention further develops detection methods based on reagent kits, enabling rapid and accurate detection. These methods can be applied to non-disease diagnostic purposes, such as detecting environmental samples for hygiene monitoring.

[0027] Optionally, the quantum dot microsphere-labeled chikungunya virus antibody and the quantum dot microsphere-labeled dengue virus antibody are mixed with the loading solution to dilute the labeled antibody by 1000-3000 times, preferably 2000 times.

[0028] In this invention, a specific dilution factor of the labeled antibody is designed during the detection process, which can further improve the detection fluorescence signal response value.

[0029] Optionally, the preparation methods of the quantum dot microsphere-labeled chikungunya virus antibody and the quantum dot microsphere-labeled dengue virus antibody include mixing and conjugating the quantum dot microspheres with the chikungunya virus antibody or the dengue virus antibody, respectively.

[0030] Optionally, the quantum dot microspheres are carboxylated quantum dot microspheres.

[0031] Optionally, prior to the mixing and coupling, the quantum dot microspheres are further mixed with a solution of 2-morpholine ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and N-hydroxysuccinimide (NHS).

[0032] Optionally, when mixing quantum dot microspheres with chikungunya virus antibody or dengue virus antibody, the amount of chikungunya virus antibody or dengue virus antibody used is 5-15 µg, preferably 8-12 µg, more preferably 9-11 µg, and even more preferably 10 µg.

[0033] Optionally, the mixing time with the sample to be tested is 1 to 5 minutes, preferably 1.5 to 2.5 minutes, and more preferably 2 minutes.

[0034] Optionally, the detection method further includes a quantitative calculation step, including detecting the fluorescence intensity value of the detection line and calculating the concentration of the virus in the sample to be tested based on a standard curve.

[0035] Optionally, the method for constructing the standard curve includes detecting samples with different known virus concentrations and recording the fluorescence intensity values ​​of the corresponding detection lines, and fitting a standard curve with the fluorescence intensity value as the ordinate and the Log value of the virus concentration as the abscissa.

[0036] Compared with the prior art, the present invention has at least the following beneficial effects:

[0037] This invention designs a reagent kit and detection method based on fluorescence immunochromatography, which has high sensitivity, strong specificity and good stability. It can realize the simultaneous quantitative detection of chikungunya virus antigen and dengue virus antigen, effectively improving the detection efficiency. Moreover, the detection operation process is simple and efficient, and the results can be reported within 15 minutes, making it suitable for rapid on-site detection. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of the Chikungunya virus / dengue virus antigen test strip. 1 is the sample pad, 2 is the absorbent paper, 3 is the nitrocellulose membrane, and 4 is the base plate.

[0039] Figure 2 The graph shows the screening results of the T1 line concentration and conjugate dilution factor for the CHIKV antigen test strip.

[0040] Figure 3 The graph shows the screening results of the T2 line concentration and conjugate dilution factor for the DENV antigen test strip.

[0041] Figure 4 The graph shows the screening results for the usage of two conjugate antibodies, CHIKV and DENV.

[0042] Figure 5 The image shows the screening results for antigen-antibody incubation time.

[0043] Figure 6 This is a graph showing the screening results of the NC membrane for the antigen test strip.

[0044] Figure 7 This is a standard curve for CHIKV antigen detection.

[0045] Figure 8 This is a standard curve for DENV antigen detection.

[0046] Figure 9 This is an image of the CHIKV antigen test strip after sample chromatography under a gel imaging system.

[0047] Figure 10 This is an image of the DENV antigen test strip after sample chromatography under a gel imaging system.

[0048] Figure 11 The image shows the test results of a simulated CHIKV serum sample.

[0049] Figure 12 The image shows the test results of a simulated serum sample for DENV. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0051] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0052] This invention designs a chikungunya virus / dengue virus antigen test strip, the structural schematic of which is shown in the figure below. Figure 1 As shown, the test strips consist of two side-by-side. Each strip is assembled from an NC membrane, a sample pad, and absorbent paper onto a base plate. Components 1-4 represent the sample pad, absorbent paper, nitrocellulose membrane, and base plate, respectively. First, a quantum dot-labeled virus-specific antibody conjugate, QBs-Ab1, is prepared by conjugating carboxylated quantum dot microspheres (QBs) with the chikungunya virus (CHIKV) specific monoclonal antibody Ab1 using an activated ester method. The NC membrane is coated with CHIKV capture antibody Ab2 (T1 line) and goat anti-mouse IgG polyclonal antibody (C line). The dengue virus (DENV) antibody conjugate, QBs-Ab3, is prepared using the same method, with the NC membrane coated with DENV capture antibody Ab4 (T2 line) and goat anti-mouse IgG polyclonal antibody (C line). When a sample containing CHIKV or DENV is mixed with the conjugate diluted with the loading solution, QBs-Ab1 or QBs-Ab3 binds to the CHIKV or DENV antigen in the sample to form a QBs-Ab1-Ag complex or a QBs-Ab3-Ag complex. The complex is then added to the sample pad and chromatography upwards to the T1 / T2 line. The capture antibody binds to the CHIKV or DENV antigen in the complex, forming a QBs-Ab1 / Ab3-Ab2 / Ab4 complex that deposits at the T1 or T2 line. The remaining complex continues to precipitate upwards, and Ab1 or Ab3 in the complex reacts with goat anti-mouse IgG and deposits at the C line. Approximately 15 minutes after sample addition, under UV light, if both the T1 / T2 and C lines show red fluorescent bands, the result is positive; if only the C line shows a fluorescent band, the result is negative; if no fluorescent band appears at the C line, the test strip is invalid. Combined with a dual-channel fluorescence immunoassay analyzer, CHIKV / DENV antigens can be quantified simultaneously.

[0053] In one embodiment of the present invention, a chikungunya virus antigen and dengue virus antigen detection kit may be provided. The kit may include the chikungunya virus / dengue virus antigen detection test strip, quantum dot microsphere-labeled chikungunya virus antibody, quantum dot microsphere-labeled dengue virus antibody, and sample loading solution, etc.

[0054] The kit preparation method includes: combining the quantum dot microsphere-labeled chikungunya virus antibody, the quantum dot microsphere-labeled dengue virus antibody, the sample loading solution, and the immunochromatographic test strip.

[0055] In another embodiment of the present invention, a method for detecting chikungunya virus antigen and dengue virus antigen is provided, using the kit described above, comprising:

[0056] The sample loading solutions of the quantum dot microsphere-labeled chikungunya virus antibody and the quantum dot microsphere-labeled dengue virus antibody were mixed to obtain the chikungunya virus sample loading solution and the dengue virus sample loading solution, respectively. These were then mixed with the samples to be tested and added to the chikungunya virus antigen test strip and the dengue virus antigen test strip, respectively. The fluorescence of the test line and the control line was observed.

[0057] The main reagents in the specific embodiments of the present invention include:

[0058] Carboxylated quantum dot microspheres: purchased from Beijing Nanokin Biotechnology Co., Ltd., model: FM610C, specification: 1mL / vial.

[0059] Mouse anti-Chikungunya virus monoclonal antibody Ab1, purchased from Shanghai Medis Medical Technology Co., Ltd., model: V3583, specification: 1mg / vial; Mouse anti-Chikungunya virus monoclonal antibody Ab2, purchased from Shanghai Medis Medical Technology Co., Ltd., model: V3585, specification: 1mg / vial.

[0060] Mouse anti-dengue virus monoclonal antibody Ab3, purchased from Changzhou Sino-American Xinxin Biotechnology Co., Ltd., model: ACT-Ab-DENV-001, specification: 1mg / vial; Mouse anti-dengue virus monoclonal antibody Ab4, purchased from Changzhou Sino-American Xinxin Biotechnology Co., Ltd., model: ACT-Ab-DENV-002, specification: 1mg / vial.

[0061] Chikungunya virus E1 & E2 antigens were purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., model: ACT-Ag-CHIKV-011, specification: 0.2mg / vial.

[0062] The dengue virus NS1 antigen was purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., model: ACT-Ag-DENV-001, specification: 0.5mg / vial.

[0063] Goat anti-mouse IgG polyclonal antibody, purchased from Changzhou Zhongmei Xinxin Biotechnology Co., Ltd., model: ACT-Ag-IgG(M)-001, specification: 2.0mg / vial.

[0064] N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Beijing Bailingwei Technology Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Beijing Innocare Technology Co., Ltd. 2-(N-morpholine)ethanesulfonic acid (MES) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Polyethylene glycol-20000 (PEG-20000), sucrose, and bovine serum albumin (BSA) were purchased from Sinopharm Chemical Reagent Co., Ltd. Anhydrous trehalose was purchased from Shanghai TCI Chemical Industry Development Co., Ltd. Tween-20 was purchased from Sigma-Aldrich, Inc., USA.

[0065] The main instruments in the specific embodiments of this invention include:

[0066] The automatic gold spraying and film drawing instrument (model: WRF-HPY001) was purchased from Haining Weierfen Automation Equipment Co., Ltd.; the microcomputer automatic chopping machine (model: ZQ2000) and CNC strip cutting machine (model: CTS300) were both purchased from Shanghai Jinbiao Biotechnology Co., Ltd.; the dry fluorescence immunoassay analyzer (model: FIC-S100) was purchased from Suzhou Hemai Precision Instrument Co., Ltd.; the fully automatic gel imaging instrument (model: Fusion FX Spectra) was produced by VILBER LOURMAT of France; and the small ultrasonic cleaner (model: JP-3800S) was purchased from Shenzhen Jiemeng Cleaning Equipment Co., Ltd.

[0067] Example 1

[0068] This embodiment describes the preparation of test strips for chikungunya virus antigen and dengue virus antigen.

[0069] Preparation process:

[0070] Two nitrocellulose membranes (NC membranes) were adhered to the substrates, and two membranes were etched. Using an automated gold-spraying etcher, CHIKV E1 & E2 protein capture antibody Ab2 (diluted to 0.8 mg / mL) and DENV NS1 protein capture antibody Ab4 (diluted to 0.8 mg / mL) were coated onto the NC membranes as detection lines (T1, T2), respectively. Goat anti-mouse IgG polyclonal antibody (diluted to 0.5 mg / mL) was coated onto the NC membranes as a control line (C line). The membranes were etched at a speed of 1 µL / cm. After etching, both plates were dried in an oven at 37°C for 2 hours. The sample pad and absorbent paper were then assembled onto the substrates and cut into 3 mm wide strips using a CNC strip cutter. These strips were then dried, protected from light, and stored for later use.

[0071] Quantum dot microsphere labeled antibody:

[0072] Chikungunya virus antibody and dengue virus antibody were coupled to the surface of quantum dot microspheres using an activated ester method. First, mix 25 μL of quantum dot (QB) microsphere (QBs) solution (1 μmol / L) with 25 μL of MES solution (20 mM, pH 6.0), then add 1 μL of EDC solution (20 mg / mL) and 1 μL of NHS solution (20 mg / mL), and activate at 37°C in the dark for 15 minutes. Centrifuge at 10000 rcf for 20 minutes, discard the supernatant, and resuspend the precipitate in 25 μL of MES solution (10 mM, pH 6.0), vortex to mix. Next, add 10 μg of CHIKV antibody / DENV antibody, and incubate at 37°C and 800 rpm in the dark for 1 hour. Add 25 μL of 10% BSA solution, vortex to mix, block at 37°C in the dark for 30 minutes, centrifuge at 8000 rcf for 15 minutes, discard the supernatant, and add 50 µL of borate buffer (5... Resuspend and wash once with 5 mM (pH 8.0) + 1% BSA solution, centrifuge at 8000 rcf for 15 minutes, and discard the supernatant; finally, add 25 µL borate buffer (5 mM, pH 8.0) + 1% BSA solution to resuspend, and obtain quantum dot microsphere-labeled chikungunya virus antibody and quantum dot microsphere-labeled dengue virus antibody, which are stored at 4℃ for later use.

[0073] Detection method:

[0074] Take an appropriate amount of the stored quantum dot-labeled CHIKV and DENV antibody conjugates and dilute them 2000-fold with loading buffer (Tris-HCl, with added Tween-20, sugar, BSA, and PEG-20000). Then, add CHIKV and DENV antigens to their respective dilutions to prepare positive samples; samples without antigens serve as negative controls. Incubate these mixtures at 37°C for 2 minutes in a dual-channel detector. Accurately drop 60 µL of the incubated sample onto the sample pads corresponding to the CHIKV and DENV detection areas on the dual-channel detection card. Finally, place the detection card into the instrument for chromatography at 37°C and read the results.

[0075] Example 2

[0076] This embodiment optimizes the detection process.

[0077] 1. Optimization of sample loading solution composition

[0078] The main influencing factors on the loading solution were analyzed, and the optimal ratio of pH, sucrose, PEG-20000, and Tween-20 was designed. Other components of the loading solution were 3% trehalose, 0.5% BSA, and an ion concentration of 0.05 mol / L. The design scheme is shown in Table 1. The same sample was tested using different loading solutions. Based on the test results, the order of influence on the total T1 and T2 signal values ​​was sucrose > PEG-20000 > pH > Tween-20. The optimal loading solution ratio was: 0.05 mol / L pH 6.5 Tris-HCl, containing 3% sucrose, 3% trehalose, 0.5% PEG-2000, 0.5% BSA, and 1% Tween-20.

[0079] Table 1

[0080]

[0081] 2. Optimization of T-line concentration and coupling agent dilution factor

[0082] For the combined test strip, the antibody concentrations for the T1 and T2 lines were set to 0.5, 0.8, and 1.0 mg / mL, respectively. During testing, the sample solution was mixed with the labeled antibody, and the diluted labeled antibody was set to 1500, 2000, and 2500 times. The combined test strip was used to test the corresponding CHIKV antigen sample (50 ng / mL) and DENV antigen sample (5 ng / mL), respectively, with no corresponding antigen sample added as a negative control. The test results are as follows: Figure 2 and 3As shown, in experiments 1-3, the conjugate was diluted 1500-fold, with T-line concentrations of 0.5, 0.8, and 1.0 mg / mL, respectively; in experiments 4-6, the conjugate was diluted 2000-fold, with T-line concentrations of 0.5, 0.8, and 1.0 mg / mL, respectively; and in experiments 7-9, the conjugate was diluted 2500-fold, with T-line concentrations of 0.5, 0.8, and 1.0 mg / mL, respectively. For both test strips, at each dilution factor, the signal value increased with increasing T-line concentration, and the increase at 0.8 mg / mL was greater than that at 1.0 mg / mL. Therefore, a T-line concentration of 0.8 mg / mL can be selected subsequently. For the CHIKV test strip, the signal value showed a trend of first increasing and then decreasing with increasing conjugate dilution factor. This may be because the conjugate aggregated at low dilution factors, thus affecting the chromatography effect. The conjugate showed the highest signal value and lowest background value at a 2000-fold dilution; therefore, a 2000-fold dilution was chosen. For the DENV test strip, the signal value decreased with increasing conjugate dilution; at a 2000-fold dilution, the background value was low, so a 2000-fold dilution was also chosen. In summary, the T-line concentration of the combined detection test strips was 0.8 mg / mL, and the dilution of both labeled viral antibodies was chosen to be 2000-fold.

[0083] 3. Amount of conjugated antibody, incubation time, and NC membrane screening.

[0084] The antibody dosage of the two quantum dot-labeled antibodies was screened, with 5, 10, and 15 µg of antibody added to the quantum dots for conjugation. The results are as follows: Figure 4 As shown, when the antibody addition amount is 10 µg, the detection signal of the combined detection strip has reached the threshold. Further increasing the antibody level results in almost no further increase in the detection signal value. Therefore, for the combined detection system, the optimal amount of conjugated antibody is 10 µg.

[0085] The incubation process before detection allows the antigen and antibody to react fully, thereby improving detection sensitivity. Incubation times of 1, 2, 4, and 5 minutes were selected, and the results were as follows: Figure 5 As shown, when the incubation time is 2 minutes, the signal values ​​of the combined test strips have all reached the threshold level. Further increasing the incubation time does not increase the signal values. Therefore, the incubation time for the detection system is set to 2 minutes.

[0086] Figure 6 To screen the detection signals of different nitrocellulose membranes, including CN140, CN95, and Millipore 135 membranes, the results showed that the CN95 membrane significantly outperformed other commercially available NC membranes, especially in the simultaneous detection of dual viruses, producing the highest signal-to-noise ratio and the largest detection signal value for both viral targets. Therefore, the CN95 membrane was selected as the nitrocellulose membrane for this detection system.

[0087] Example 3

[0088] This embodiment examines the detection performance of the test strip.

[0089] 1. Sensitivity

[0090] After diluting the corresponding conjugates using the selected optimal loading solution system, different concentrations of CHIKV antigen (0.01, 0.05, 0.1, 0.5, 5.0, 50, 100, 500, 1000, 2000 ng / mL) and DENV antigen (0.001, 0.005, 0.01, 0.05, 0.5, 2.5, 10, 25, 100, 250 ng / mL) were added, followed by incubation and detection. A standard curve was plotted based on the detected signal values. The CHIKV standard curve is shown below. Figure 7 As shown, the equation of the curve is: y = 378.9 + (17267 - 378.9) / (1 + 10^((4.950 - x) × 1.656)), R 2 =0.9954. The calculated LOD is 50 pg / mL, with a visual LOD of 100 pg / mL. The DENV standard curve is as follows: Figure 8 As shown, the equation of the curve is: y = 333.6 + (17435 - 333.6) / (1 + 10^((4.436 - x) × 1.154)), R 2 =0.9956. The calculated LOD is 2 pg / mL, with a visual LOD of 10 pg / mL. This indicates that the test strip of this invention possesses good detection sensitivity.

[0091] 2. Specificity

[0092] The assembled CHIKV / DENV antigen combined test strip was used to detect CHIKV antigen, DENV antigen, yellow fever virus (YFV) antigen, and Zika virus (ZIKV) antigen at a concentration of 500 ng / mL. After chromatography for 15 min, each test was performed in triplicate. The results are as follows: Figure 9 As shown, the CHIKV antigen test result was positive, while the results of other samples were negative. The assembled combined test strip was used to detect DENV antigen, CHIKV antigen, yellow fever virus (YFV) antigen, and Zika virus (ZIKV) antigen at a concentration of 100 ng / mL. After chromatography for 15 min, each test was performed in triplicate. The results are shown below. Figure 10 As shown, it can be observed that, except for a positive result for the DENV antigen test, all other sample test results were negative. This indicates that the prepared combined test strip has good specificity and no cross-reactivity with other related pathogens.

[0093] 3. Serum simulated sample test

[0094] This embodiment uses simulated samples to evaluate the test strip performance. Ten clinically negative serum samples were mixed. The CHIKV antibody conjugate was diluted 2000 times with the loading solution, and the mixed serum was added to achieve serum dilutions of 5, 10, and 20 times. Then, CHIKV antigen was added to a final concentration of 50 ng / mL. The DENV antibody conjugate was diluted 2000 times with the loading solution, and the mixed serum was added to achieve serum dilutions of 5, 10, and 20 times. Then, DENV antigen was added to a final concentration of 5 ng / mL. A control group without added serum was also prepared. The combined test strip was used to test the above samples, and the test results are as follows. Figure 11 and Figure 12 As shown in the results, the test strips of this invention can effectively detect viral antigens in serum. The detection signal value of the two test strips increases the most when the serum dilution factor is 10 times. Further increasing the serum dilution factor does not significantly increase the signal value. The signal value of the 20-fold dilution is basically close to the detection result of the control group without serum. Therefore, the optimal serum dilution factor for the combined test strips is 10 times.

[0095] 4. Quantitative detection of serum simulated samples

[0096] The prepared combined test strip was used to quantitatively detect serum simulants diluted 10-fold. The concentrations of the CHIKV simulants were set to 0.1, 50, and 250 ng / mL; the concentrations of the DENV simulants were set to 0.01, 5, and 25 ng / mL. The sample concentrations were calculated according to the standard curve, and the results are shown in Table 2. It can be seen that the quantitative concentrations of CHIKV and DENV in the serum simulants of the present invention are basically consistent with the actual concentrations, and the recovery rates are between 95.40% and 108.00%, showing the accuracy of the quantitative results.

[0097] Table 2

[0098]

[0099] In summary, this invention utilizes fluorescent quantum dot microspheres as markers to prepare chromatographic strips, kits, and detection methods for the combined detection of chikungunya virus antigen and dengue virus antigen. This enables the combined detection of chikungunya virus antigen and dengue virus antigen, and the results can be determined within 15 minutes using ultraviolet light. Furthermore, the detection of the fluorescence signal value can be combined to achieve viral quantification, making it suitable for rapid on-site detection. The detection exhibits good sensitivity, specificity, and stability, providing a new approach for the prevention, control, and treatment of chikungunya and dengue viruses.

[0100] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A detection kit for chikungunya virus antigen and dengue virus antigen, characterized in that, The kit includes quantum dot microsphere-labeled chikungunya virus antibody, quantum dot microsphere-labeled dengue virus antibody, sample loading solution, and immunochromatographic test strip; The immunochromatographic test strip consists of a chikungunya virus antigen test strip and a dengue virus antigen test strip arranged side by side. The detection line of the Chikungunya virus antigen test strip is coated with Chikungunya virus capture antibody, and the control line is coated with goat anti-mouse IgG polyclonal antibody. The dengue virus antigen test strip has a detection line coated with dengue virus capture antibodies and a control line coated with goat anti-mouse IgG polyclonal antibodies.

2. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 1, characterized in that, The loading solution contains Tris-HCl, sucrose, trehalose, polyethylene glycol, bovine serum albumin, and Tween.

3. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 2, characterized in that, The polyethylene glycol includes PEG-20000; The Tween includes Tween-20.

4. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 2, characterized in that, The loading solution contains 0.025~0.15 mol / L Tris-HCl, 0.5~6 wt% sucrose, 0.5~6 wt% trehalose, 0.25~1 wt% polyethylene glycol, 0.1~1 wt% bovine serum albumin and 0.5~3 wt% Tween; The pH of the loading solution is 5.5~8.

0.

5. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 1, characterized in that, The Chikungunya virus antigen test strip and the dengue virus antigen test strip each independently include a base plate, a sample pad, a detection membrane, and absorbent paper, and the sample pad, the detection membrane, and the absorbent paper are sequentially connected and arranged on the base plate; The detection membrane includes a nitrocellulose membrane.

6. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 1, characterized in that, The detection strip of the Chikungunya virus antigen test strip contains a Chikungunya virus capture antibody concentration of 0.5~1.5 mg / mL. The dengue virus antigen test strip contains dengue virus capture antibody at a concentration of 0.5–1.5 mg / mL in the detection strip.

7. The detection kit for chikungunya virus antigen and dengue virus antigen according to claim 1, characterized in that, The quantum dot microsphere-labeled chikungunya virus antibody and the chikungunya virus capture antibody bind to the chikungunya virus at different sites, and the quantum dot microsphere-labeled dengue virus antibody and the dengue virus capture antibody bind to the dengue virus at different sites.

8. A method for preparing the detection kit for chikungunya virus antigen and dengue virus antigen according to any one of claims 1-7, characterized in that, The preparation method includes: combining the quantum dot microsphere-labeled chikungunya virus antibody, the quantum dot microsphere-labeled dengue virus antibody, the sample loading solution, and the immunochromatographic test strip.

9. A method for detecting chikungunya virus antigen and dengue virus antigen for purposes other than disease diagnosis and / or treatment, characterized in that, The detection method utilizes the detection kit for chikungunya virus antigen and dengue virus antigen as described in any one of claims 1-7, including: The quantum dot microsphere-labeled Chikungunya virus antibody and the quantum dot microsphere-labeled dengue virus antibody were mixed with the sample loading solution to obtain Chikungunya virus loading solution and dengue virus loading solution, respectively. These were then mixed with the samples to be tested and added to the Chikungunya virus antigen test strip and the dengue virus antigen test strip, respectively. The fluorescence of the test line and the control line was observed.

10. The method for detecting chikungunya virus antigen and dengue virus antigen for non-disease diagnosis and / or treatment purposes according to claim 9, characterized in that, The detection method also includes a quantitative calculation step, which includes detecting the fluorescence intensity value of the detection line and calculating the concentration of the virus in the sample to be tested based on a standard curve; The method for constructing the standard curve includes detecting samples with different known virus concentrations and recording the fluorescence intensity values ​​of the corresponding detection lines. The standard curve is then constructed by fitting the fluorescence intensity values ​​on the ordinate and the log value of the virus concentration on the abscissa.

Images