An infectious spleen and kidney necrosis virus double-channel double-target detection test strip and a preparation method and application thereof

Abstract

The application relates to the technical field of biology, and particularly discloses a double-channel double-target detection test strip for a spleen and kidney necrosis virus and a preparation method and application thereof. The double-channel double-target detection test strip for the spleen and kidney necrosis virus provided by the application is provided with two independent chromatographic channels, and each of the two independent chromatographic channels contains a sample pad, a gold label pad, a nitrocellulose membrane containing a detection line T and a quality control line C and a water absorption pad in sequence. The double-channel double-target detection test strip for the spleen and kidney necrosis virus provided by the application has good specificity for ISKNV and has no obvious cross reaction with SDDV, SGIV, MRV, NNV and SCRV. The detection limit of a VP023 detection test strip is 10 6 copies / μL, and the detection limit of a VP101 detection test strip is 10 4 copies / μL. The detection test strip can be used for ISKNV detection in liver, spleen, kidney and blood samples.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a dual-channel dual-target test strip for the detection of infectious spleen and kidney necrosis virus, its preparation method, and its application. Background Technology

[0002] Infectious spleen and kidney necrosis virus (ISKNV) belongs to the Iridoviridae family ( Iridoviridae ), genus Mammocyte Virus ( Megalocytivirus ISKNV can infect various types of fish. Infection with ISKNV can cause tissue lesions and death, therefore it is necessary to detect ISKNV in samples.

[0003] Currently, the main methods for detecting ISKNV include virus isolation and culture, conventional PCR, quantitative real-time PCR, isothermal amplification, and digital PCR. While these methods can be used for ISKNV detection, they typically require nucleic acid extraction, amplification reactions, or instrumental detection, involving numerous operational steps. For scenarios requiring initial sample screening or rapid interpretation, existing detection methods still have limitations in terms of operational procedures and detection time.

[0004] Colloidal gold immunochromatographic test strips allow for interpretation of colorimetric results, offering fewer operational steps and faster detection speed. For ISKNV, the expression time of related proteins varies at different infection stages. If the detection system simultaneously includes detection targets for proteins related to different infection stages, colorimetric results for different proteins can be obtained while detecting the ISKNV antigen.

[0005] Therefore, there is a need for a colloidal gold immunochromatographic test strip for detecting ISKNV. This test strip should be able to simultaneously detect both early non-structural proteins and late-stage related proteins to achieve dual-target detection of the ISKNV antigen. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art by providing a dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus (ISKNV), its preparation method, and its application. The test strip provided in this application is constructed based on colloidal gold immunochromatography technology, using early non-structural proteins and late-stage related proteins of ISKNV as detection targets. It enables rapid and visual detection of ISKNV and allows for preliminary assessment of the infection status based on the detection results of different targets.

[0007] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a dual-channel dual-target test strip for infectious spleen and kidney necrosis virus, which has two independent chromatography channels set on the same substrate. Each of the two independent chromatography channels contains a sample pad, a gold label pad, a nitrocellulose membrane containing a detection line T and a control line C, and an absorbent pad in sequence. The sample pad, gold label pad, nitrocellulose membrane containing detection line T and control line C, and absorbent pad are sequentially overlapped on the base plate; the detection line T is located at the end near the conjugate pad, and the control line C is located at the end near the absorbent pad. The gold-labeled pads of the two independent chromatography channels were coated with labeled antibodies for early non-structural proteins and late-stage related proteins of infectious spleen and kidney necrosis virus, respectively. The detection lines T of the two independent chromatography channels are coated with antibodies for detecting early non-structural proteins and late-stage related proteins of infectious spleen and kidney necrosis virus, respectively.

[0008] In some specific embodiments, the infection status of infectious spleen and kidney necrosis virus in the sample is preliminarily determined based on the color development of the detection line T.

[0009] In some specific embodiments, the interpretation logic is as follows: only early non-structural protein positive = early indication; only late related protein positive = particle related / late; double positive for both early non-structural protein and late related protein = strong activity; only C positive = negative / below the detection limit; no C line = invalid.

[0010] In the technical solution of this application, the two independent chromatography channels of the ISKNV dual-channel dual-target test strip provided by this application detect early non-structural proteins and late-related proteins respectively. The test strip can be used to detect ISKNV antigen in fish samples, and the infection status can be preliminarily judged according to the color development of the dual detection lines. The early non-structural proteins are used for early infection-related detection, and the late-related proteins are used for mid-to-late-stage infection-related detection.

[0011] The results showed that the test strip had good specificity for ISKNV and no significant cross-reactivity with SDDV, SGIV, MRV, NNV, and SCRV; the detection limit of the test strip for early non-structural proteins was 10. 6 The detection limit for the test strip for late-stage related proteins is 10 copies / μL. 4 This test strip can be used to detect ISKNV in liver, spleen, kidney, and blood samples. (copies / μL)

[0012] In a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the early non-structural protein includes the early non-structural protein VP023; and the late-related protein includes the late-related protein VP101.

[0013] This application uses ISKNV proteins (VP023 and VP101) at different infection stages as detection targets, which can simultaneously obtain the detection results of two viral proteins and be used for preliminary judgment of infection status.

[0014] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the amino acid sequence of the early non-structural protein VP023 is shown in SEQ ID NO: 1; The amino acid sequence of the late-stage related protein VP101 is shown in SEQ ID NO: 2.

[0015] In a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the labeling antibody for the early non-structural protein includes the monoclonal antibody ISKNV-3C5.

[0016] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the antibody for detecting early non-structural proteins includes the monoclonal antibody ISKNV-2K10.

[0017] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the marker antibody for late-related proteins includes the monoclonal antibody ISKNV-3D15.

[0018] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the antibody for detecting late-related proteins includes the monoclonal antibody ISKNV-1B19.

[0019] The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-3C5 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-2K10 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-3D15 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-1B19 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0020] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the coating concentration of the detection line T of the two independent chromatography channels is 1-2 mg / mL, preferably 1 mg / mL; The control line C of the two independent chromatography channels is coated with goat anti-mouse IgG at a concentration of 0.5-1 mg / mL, preferably 0.5 mg / mL.

[0021] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the labeled antibodies for early non-structural proteins or late-related proteins are labeled with colloidal gold particles with a particle size of 20-40 nm.

[0022] As a preferred embodiment of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus described in this application, the labeling concentration of the monoclonal antibody ISKNV-3C5 is 17 μg / mL, and the pH of the system during labeling is 8.3-8.5; the labeling concentration of the monoclonal antibody ISKNV-3D15 is 13 μg / mL, and the pH of the system during labeling is 8.8-9.0.

[0023] This application also provides a method for preparing the aforementioned dual-channel dual-target test strip for infectious spleen and kidney necrosis virus, comprising the following steps: (1) Monoclonal antibodies ISKNV-3C5 and ISKNV-3D15 were conjugated with colloidal gold particles to obtain labeled antibodies for early non-structural proteins and labeled antibodies for late-stage related proteins. (2) The labeled antibodies for early non-structural proteins and late related proteins obtained in step (1) were coated onto the gold pads corresponding to the two chromatography channels, respectively. (3) The detection antibodies for early non-structural proteins and late related proteins are respectively coated onto the nitrocellulose membranes corresponding to the two chromatography channels to form the detection line T for early non-structural proteins and the detection line T for late related proteins, and quality control lines are set. (4) Assemble the sample pad, gold label pad, nitrocellulose membrane and absorbent pad in sequence to obtain the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus.

[0024] This application uses colloidal gold immunochromatography, which is a simple and quick detection method that does not require complex instruments or equipment.

[0025] The test strips developed in this application have good specificity for ISKNV and show no significant cross-reactivity with common fish viruses such as SDDV, SGIV, MRV, NNV, and SCRV.

[0026] Furthermore, the detection limit of the VP101 test strip can reach 10. 4The detection limit of the VP023 test strip can reach 10 copies / μL. 6 copies / μL.

[0027] This application also provides the application of the above-mentioned dual-channel dual-target test strip for infectious spleen and kidney necrosis virus in the preparation of antigen products for detecting infectious spleen and kidney necrosis virus in fish samples.

[0028] As a preferred embodiment of the application described in this application, the fish sample includes at least one of a liver sample, a spleen sample, a kidney sample, and a blood sample.

[0029] The test strips provided in this application are applicable to the detection of various fish samples, especially to the rapid detection of ISKNV in tissue and blood samples; in this application, VP023 can be used for early infection identification, VP101 can be used for mid-to-late stage infection detection, and the dual-target combined detection can be used for preliminary judgment of infection status.

[0030] This application also provides the method of using the above-mentioned dual-channel dual-target test strip for infectious spleen and kidney necrosis virus. For tissue samples, add 4-6 mL of PBS containing 0.05-0.2% Triton-100 per 1 g of tissue, homogenize, and centrifuge to collect the supernatant. For blood samples, dilute PBS containing 0.05-0.2% Triton-100 at a 1:1 ratio as the test sample. The sample volume added each time is 60-100 μL, and the reading time is 10-20 min.

[0031] Compared with the prior art, this application has the following beneficial effects: This application provides a dual-channel, dual-target test strip for the detection of infectious spleen and kidney necrosis virus (ISKNV), its preparation method, and its application. The two independent chromatographic channels of the ISKNV dual-channel, dual-target test strip provided in this application detect early non-structural proteins and late-stage related proteins, respectively. This test strip can be used to detect ISKNV antigen in fish samples, and the infection status can be preliminarily judged based on the color development of the dual detection lines. Early non-structural proteins are used for early infection-related detection, while late-stage related proteins are used for mid-to-late-stage infection-related detection. The test strip provided in this application has good specificity for ISKNV and shows no significant cross-reactivity with common fish viruses such as SDDV, SGIV, MRV, NNV, and SCRV. The test strip is suitable for detection in various fish samples, especially for rapid detection of ISKNV in tissue and blood samples. Attached Figure Description

[0032] Figure 1 This is a schematic diagram illustrating the structure and interpretation of a dual-channel, dual-target test strip for the detection of infectious spleen and kidney necrosis virus; among which, Figure 1A is a schematic diagram of the basic components of a test strip. Figure 1 B is a schematic diagram of the dual-channel detection card structure. Figure 1 C is a schematic diagram illustrating the interpretation of different test results; Figure 2 The temporal expression characteristics of vp023 and VP101 as detection targets when ISKNV infects MFF-1 cells; among them, Figure 2 A represents the immunofluorescence results at different time points after ISKNV infection of MFF-1 cells. Figure 2 B represents the Western blot results at different time points after ISKNV infection of MFF-1 cells. Figure 2 C is the expression trend graph of vp023 and VP101. Figure 2 D represents the Western blot results of vp023 and VP101 in the supernatant and precipitate fraction of purified ISKNV after SDS treatment. Figure 2 D indicates that VP101 was mainly found in the precipitated fraction, while vp023 was not found in the purified virus particles. Figure 2 E is a diagram showing the localization results of vp023 and VP101 in infected cells. Figure 2 E indicates that VP101 is mainly located in the viral assembly region, while vp023 is mainly located in the infected cell membrane. Figure 3 Representative identification results of pairing and screening different candidate monoclonal antibodies using the checkerboard method; the left side shows the identification results of candidate antibody pairing in the VP023 detection channel, and the right side shows the identification results of candidate antibody pairing in the VP101 detection channel. Figure 4 The graph shows the sensitivity and specificity test results for the VP023 and VP101 test strips; among them, Figure 4 A represents the sensitivity result of the VP023 test strip. Figure 4 B represents the sensitivity result of the VP101 test strip. Figure 4 C represents the specificity result of the VP023 test strip. Figure 4 D represents the specificity result of the VP101 test strip; Figure 5 The image shows the detection results of the dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus in infected tissues and different sample types; among them, Figure 5 A represents the immunofluorescence detection results of spleen tissue after artificial infection of mandarin fish. Figure 5 B represents the test strip results at different time points after artificially infecting mandarin fish. Figure 5 C represents the test results after gradient dilution of the infected tissue homogenate. Figure 5 D represents the test results in liver, spleen, kidney, and blood samples. Detailed Implementation

[0033] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0034] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified, and the raw materials used in each parallel experiment are the same.

[0035] Example 1: Selection of Targets for VP023 and VP101 Detection 1. To construct a rapid detection system that reflects different stages of ISKNV infection, this application selected the early non-structural protein vp023 and the late structural protein VP101 of ISKNV as detection targets. Immunofluorescence detection and Western blot analysis of MFF-1 cells infected with ISKNV at different time points (0h, 4h, 8h, 12h, 24h, 36h, 48h, 72h) showed that vp023 signal could be detected as early as 12h post-infection, while VP101 signal appeared at 36h post-infection, indicating that vp023 is expressed early in cell infection, while VP101 is expressed more significantly at a later stage. The relevant results are shown in [reference needed]. Figure 2 A and Figure 2 B. Figure 2 A represents the immunofluorescence results at different time points after ISKNV infection of MFF-1 cells. Figure 2 B represents the Western blot results at different time points after ISKNV infection of MFF-1 cells.

[0036] 2. Tissue sample testing was performed after artificially infecting mandarin fish. The specific steps were as follows: Mandarin fish were used as the infection target, and artificial infection was carried out by intraperitoneal injection at a dose of 10^8.2 copies / fish. Samples were taken on days 1, 3, 5, and 7 post-infection, with 3 fish taken at each time point. Immunofluorescence was used as a control.

[0037] The results showed that VP023 showed a positive signal 3 days after infection, while VP101 showed a significant positive signal 5 days after infection. (See attached results). Figure 5 A, 5B. Figure 5 A represents the immunofluorescence detection results of spleen tissue after artificial infection of mandarin fish. Figure 5 B represents the test strip results at different time points after artificially infecting mandarin fish.

[0038] 3. Infect MFF-1 cells with ISKNV. After complete cytopathic effects appear, collect the sample and centrifuge at 8000 ×g, 4 °C for 40 min to remove cell debris, retaining the supernatant. Then, ultracentrifuge the supernatant at 80,000 ×g, 4 °C for 90 min, discard the supernatant, and resuspend the pellet in PBS to obtain the crude virus extract. Spread the crude virus extract onto a PBS buffer containing 35% (w / v) sucrose and ultracentrifuge at 150,000 ×g, 4 °C for 1 h, discard the supernatant, and resuspend the pellet in PBS. Then, load the sample onto the top of a 30%–60% (w / v) sucrose density gradient and centrifuge at 150,000 ×g, 4 °C for 1 h. Collect the milky white band visible in the middle of the gradient, mix with PBS, and centrifuge again at 150,000 ×g, 4 °C for 1 h for purification. Finally, discard the supernatant, resuspend the virus pellet in PBS, and store at -80 °C for later use. The purified ISKNV sample was treated with 1% SDS at room temperature for 90 seconds, then centrifuged at 15,000×g for 10 minutes to separate the supernatant and precipitate, which were used for subsequent detection.

[0039] The results showed that VP101 was mainly present in the precipitate, while vp023 was not detected in the purified virus particles, indicating that VP101 mainly exists as a structural protein in the virus particles, while vp023 is not a structural component of the virus particles.

[0040] Figure 2 C is the expression trend graph of vp023 and VP101. Figure 2 D represents the Western blot results of vp023 and VP101 in the supernatant and precipitate fraction of purified ISKNV after SDS treatment. Figure 2 E represents the localization results of vp023 and VP101 in infected cells. Figure 2 D indicates that VP101 mainly appeared in the precipitate, while vp023 was not found in the purified virus particles; Figure 2 E indicates that VP101 is mainly located in the viral assembly region, while vp023 is mainly located in the infected cell membrane.

[0041] The immunofluorescence localization results showed that VP101 was mainly distributed in the viral assembly region, while vp023 was mainly located in the infected cell membrane, suggesting that vp023 is more likely to be involved in the cell membrane modification process related to infection. These results indicate that vp023 is more suitable as a target for early infection-related detection, while VP101 is more suitable as a target for mid-to-late stage infection-related detection.

[0042] Example 2: Preparation of Antigen The antigen used to prepare the anti-vp023 monoclonal antibody was the N-terminal fragment of the early non-structural protein vp023, which bypassed the signal peptide region and subsequent laminin homologous region of the vp023 protein and was fused with MBP for expression. The antigen used to prepare the anti-VP101 monoclonal antibody was the full-length late-related protein VP101, with a coding sequence length of 516 nucleotides. Both vp023 and VP101 antigens were prepared using prokaryotic expression of recombinant proteins, with the expression vector pMAL-c2X and the expression host BL21. The expression products were purified using MBP affinity purification (GE Healthcare Life Sciences).

[0043] The amino acid sequence of the N-terminal fragment of the early non-structural protein vp023 is shown in SEQ ID NO: 1; The amino acid sequence of the late-stage related protein VP101 is shown in SEQ ID NO: 2.

[0044] Example 3: Obtaining and Screening Monoclonal Antibodies Monoclonal antibodies were prepared using the N-terminal fragment of the early non-structural protein vp023 and the late-related protein VP101 obtained above as immunogens.

[0045] The specific steps are as follows: The purified protein solution was diluted to 1 mg / mL and mixed 1:1 with a 3-week standard mouse monoclonal antibody adjuvant (Shanghai Yuanye Biotechnology). Balb / C mice were immunized via intramuscular injection in the leg, with three mice immunized at a dose of 50 μg per mouse. A total of three immunizations were performed. The second immunization was administered 21 days after the first, and a booster immunization of 100 μg protein was administered intraperitoneally 14 days after the second immunization. Logarithmic growth phase SP2 / 0 cells were collected, and mouse spleens were aseptically isolated and ground to prepare a spleen cell suspension. This suspension was mixed with SP2 / 0 cells and centrifuged at 1500 rpm for 5 min. After resuspending the cells, PEG 1450 was added in a 37℃ water bath for cell fusion. Serum-free DMEM was added to terminate the reaction, and the cells were centrifuged again at 1500 rpm for 5 min before resuspending in DMEM. Balb / C mouse thymus feeder cells were added and mixed into methylcellulose semi-solid medium (DMEM medium containing 2% methylcellulose (Sigma-Aldrich), 10% FBS (Gibco), and a final concentration of 1x HAT (Invitrogen)). The medium was then incubated at 37°C in a 5% CO2 incubator. After 10 days, single colonies were picked and expanded into 96-well plates. ELISA was performed after 3 days, and positive cells were further cultured and cryopreserved.

[0046] Subcloning completed monoclonal positive cell expansion culture. Three days prior to the procedure, healthy BALB / c mice were intraperitoneally injected with 100 µL of Freund's incomplete adjuvant (Sigma-Aldrich). Three days later, hybridoma cells were inoculated into the peritoneal cavity of each Balb / c mouse at a dose of 0.6 × 10⁶ cells / mc². 6 Cells. Approximately one week after injection, ascites began to form in the mouse peritoneum. The mice were observed daily, and when sufficient ascites was collected, they were euthanized with CO2. The ascites was collected by inserting a needle into the peritoneal cavity, centrifuged, aliquoted, and stored at -80 °C. The ascites antibody was purified using Protein A Agarose (Fast Flow, for antibody purification) reagents (Beyotime Biotechnology Co., Ltd.) according to the instruction manual.

[0047] After screening, four monoclonal antibodies were obtained that can be used for colloidal gold immunochromatographic detection. Among them, ISKNV-3C5 and ISKNV-2K10 were used for VP023 detection, and ISKNV-3D15 and ISKNV-1B19 were used for VP101 detection.

[0048] ISKNV-3C5 was used as the labeling antibody for the vp023 detection unit, and ISKNV-2K10 was used as the detection antibody for the vp023 detection unit; ISKNV-3D15 was used as the labeling antibody for the VP101 detection unit, and ISKNV-1B19 was used as the detection antibody for the VP101 detection unit.

[0049] All four monoclonal antibodies were derived from hybridoma ascites and purified with Protein A before use. Among them, ISKNV-3C5 is the IgG1 subtype, while ISKNV-2K10, ISKNV-3D15, and ISKNV-1B19 are the IgG2a subtype. The light chain of all four antibodies is the κ chain.

[0050] The amino acid sequence of the heavy chain variable region of ISKNV-3C5 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; the amino acid sequence of the heavy chain variable region of ISKNV-2K10 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of ISKNV-3D15 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; the amino acid sequence of the heavy chain variable region of ISKNV-1B19 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10.

[0051] Example 4: Preparation of colloidal gold-labeled antibodies Commercially available 20 nm colloidal gold particles (Shanghai Jinbiao Biotechnology Co., Ltd.) were used as labeling carriers. Monoclonal antibodies ISKNV-3C5 and ISKNV-3D15 were conjugated to the colloidal gold particles, respectively. The labeling concentration of ISKNV-3C5 was 17 μg / mL, and the pH of the system during labeling was 8.3; the labeling concentration of ISKNV-3D15 was 13 μg / mL, and the pH of the system during labeling was 8.8.

[0052] After coupling, blocking buffer was added for blocking. The blocking buffer consisted of 3% BSA, 3% sucrose, and 0.05% Tween-20, and the blocking time was 15 min. After blocking, the mixture was centrifuged at 4℃ and 10,000 rpm for 30 min. The supernatant was discarded, and the mixture was resuspended in 20 mM PBS, pH 7.4, containing 3% BSA, 3% sucrose, and 0.05% Tween-20. The final resuspending volume was 1 / 10 of the original system volume.

[0053] Example 5: Assembly of a dual-channel, dual-target test strip for the detection of infectious spleen and kidney necrosis virus. This embodiment provides a dual-channel dual-target test strip for infectious spleen and kidney necrosis virus. Two independent chromatography channels are set on the same substrate. Each of the two independent chromatography channels contains a sample pad, a gold label pad, a nitrocellulose membrane containing a detection line T and a control line C, and an absorbent pad in sequence. The sample pad, gold label pad, nitrocellulose membrane containing detection line T and control line C, and absorbent pad are sequentially overlapped on the base plate; the detection line T is located at the end near the conjugate pad, and the control line C is located at the end near the absorbent pad. The gold-labeled pads of the two independent chromatography channels were coated with marker antibodies (monoclonal antibody ISKNV-3C5 and monoclonal antibody ISKNV-3D15) for the early non-structural proteins and late-related proteins of infectious spleen and kidney necrosis virus, respectively. The detection lines T of the two independent chromatography channels are coated with antibodies (monoclonal antibody ISKNV-2K10 and monoclonal antibody ISKNV-1B19) to detect early non-structural proteins and late-stage related proteins of infectious spleen and kidney necrosis virus, respectively. Their structural diagrams are shown below. Figure 1 A and Figure 1 As shown in B.

[0054] The nitrocellulose membrane is CN140 membrane, and the material of the gold standard pad is VL68.

[0055] The sample pad treatment solution consists of borate buffer, pH 7.4-7.6, containing 0.1% Tween-20, 1% BSA, and 0.1 M NaCl. The glass fiber membrane or polyester membrane is immersed in the sample pad treatment solution, then removed and dried at 37°C for 2-4 hours.

[0056] The treatment solution for the gold-labeled pads consists of borate buffer solution with a pH of 7.4-7.6, containing 3% sucrose, 0.5% PEG 20000, 1% BSA, and 0.1% Tween-20. The glass fiber membrane or polyester membrane is immersed in the gold-labeled pad treatment solution, then removed and dried at 37°C for 2-4 hours.

[0057] Colloidal gold-labeled ISKNV-3C5 and ISKNV-3D15 were coated onto their respective gold-labeled pads. ISKNV-2K10 and ISKNV-1B19 were respectively placed on nitrocellulose membranes to form the VP023 detection line T and VP101 detection line T, with a coating concentration of 1 mg / mL for both. The control line was coated with goat anti-mouse IgG at a concentration of 0.5 mg / mL.

[0058] The sample pad, gold label pad, nitrocellulose membrane, and absorbent pad were assembled sequentially in the direction of chromatography to obtain a dual-channel dual-target test strip for infectious spleen and kidney necrosis virus.

[0059] Example 6: The effect of different antibody pairings on the performance of test strips To screen the optimal monoclonal antibody pairings suitable for dual-channel dual-target test strips for infectious spleen and kidney necrosis virus, a checkerboard method was used to systematically screen candidate marker antibodies and candidate detection antibodies while keeping other preparation conditions consistent. The differences in colorimetric effect, sensitivity, and specificity of different antibody pairings were compared.

[0060] In the VP023 detection channel, six candidate anti-VP023 monoclonal antibodies were selected as labeling antibodies and detection antibodies, respectively, and combined in pairs to construct an antibody pairing system. Similarly, in the VP101 detection channel, six candidate anti-VP101 monoclonal antibodies were selected as labeling antibodies and detection antibodies, respectively, and combined in pairs to construct an antibody pairing system. Test strips were prepared separately for each combination according to the method described in this application, with different labeling antibodies arranged horizontally and different detection antibodies arranged vertically. The antibody pairing screening results for the VP023 detection channel are shown in Table 1, and the antibody pairing screening results for the VP101 detection channel are shown in Table 2. The results of the one-to-one corresponding representative recognition bands are shown in Table 2. Figure 3 .

[0061] The results showed that there were significant differences in the detection performance between different monoclonal antibody pairs. Some combinations showed weak detection line color development, high background, unclear bands, or poor repeatability.

[0062] According to Table 1 and Figure 3 It is evident that in the VP023 detection channel, using monoclonal antibody ISKNV-3C5 as the labeling antibody and monoclonal antibody ISKNV-2K10 as the detection antibody resulted in the clearest detection line development, the lowest background, and stable control lines, demonstrating superior overall detection performance compared to other candidate antibody combinations; according to Table 2 and Figure 3 It is evident that in the VP101 detection channel, using monoclonal antibody ISKNV-3D15 as the labeling antibody and monoclonal antibody ISKNV-1B19 as the detection antibody resulted in the strongest color development, clearest bands, and best repeatability, demonstrating superior overall detection performance compared to other candidate antibody combinations. These results indicate that different monoclonal antibody pairings significantly impact the color development, sensitivity, and specificity of the test strip. ISKNV-3C5 / ISKNV-2K10 and ISKNV-3D15 / ISKNV-1B19 are the optimal antibody pairings for the VP023 and VP101 detection channels, respectively.

[0063] Table 1. VP023 detection monoclonal antibody pairing test Table 2 Vp101 detection monoclonal antibody pairing test Example 7: Sample Processing and Detection Methods Tissue samples from the liver, spleen, and kidneys were collected. 1 g of tissue was homogenized in 5 mL of PBS containing 0.1% Triton-100, centrifuged, and the supernatant was used as the test sample. Blood samples were diluted 1:1 with PBS containing 0.1% Triton-100 as the test sample.

[0064] During the test, 60 μL of sample was added each time, and the results were interpreted after 15 minutes. The interpretation method for the test results is as follows: Figure 1 As shown in C.

[0065] (1) The quality control line C is colored, and both the vp023 detection line T and the VP101 detection line T are colored, which indicates that ISKNV is positive, suggesting that vp023 and VP101 related antigen signals are present in the sample at the same time; (2) The control line C showed color, but only the vp023 detection line T showed color, which was determined to be ISKNV positive, indicating that there was vp023 related antigen signal in the sample, which is an early infection related test result; (3) The control line C showed color, but only the VP101 detection line T showed color, which was determined to be ISKNV positive, indicating that there was a VP101-related antigen signal in the sample. Since VP101 mainly exists as a structural protein in the virus particles, while vp023 was not found in the purified virus particles, this result suggests that when the sample is a blood sample, there may be a VP101 signal mainly composed of virus particle-related antigens in the sample. (4) The quality control line C showed color, while the test lines T for vp023 and VP101 did not show color, indicating a negative result; (5) If the quality control line C does not develop color, it is considered an invalid result.

[0066] Example 8: Sensitivity Detection The standard used for sensitivity testing was viral solution, with copies / μL calculated by absolute quantitative PCR according to the standard curve. The viral solution was serially diluted 10-fold using PBS as the diluent, with each dilution repeated 5 times. The limit of detection was determined by the concentration corresponding to the weakest visible positive line.

[0067] The results showed that the detection limit of the VP023 test strip was 10. 6 The detection limit of the VP101 test strip is 10 copies / μL. 4 copies / μL, such as Figure 4 A and Figure 4 As shown in B.

[0068] Example 9, Specificity Detection ISKNV virus stock solution (from reference 1), scaly disease virus (SDDV) stock solution (from reference 2), grouper iridovirus (GIV) stock solution (from reference 3), mandarin fish frog virus (MRV) stock solution (from reference 4), nerve necrosis virus (NNV) stock solution (from reference 5), mandarin fish rhabdovirus (SCRV) stock solution (from reference 6), and a negative control were tested. All six viruses were strains preserved in our laboratory, initially isolated from diseased fish tissue samples and stored at -80℃. SDDV, SGIV, MRV, and SCRV were virus stock solutions obtained from MFF-1 cell culture, NNV was a virus stock solution obtained from SSN-1 cell culture, and the negative control was MFF-1 cell lysate. Each sample was tested three times.

[0069] The results showed that the test strip only showed a positive signal for ISKNV samples, and no significant cross-reactivity was observed for SDDV, SGIV, MRV, NNV, SCRV, and the negative control. Figure 4 C and Figure 4 D.

[0070] 1. Dong CF, Weng SP, Shi XJ, Xu XP, Shi N, He JG.2008. Development of a mandarin fish fry cell line suitable for the study of infectious spleen andkidney necrosis virus (ISKNV). Virus Research 135:273-281. 2. Fu Y, Li Y, Fu W, Su H, Zhang L, Huang C, Weng S, Yu F, He J, DongC.2021. Scale Drop Disease Virus Associated Yellowfin Seabream (Acanthopagruslatus) Ascites Diseases, Zhuhai, Guangdong, Southern China: The FirstDescription. Viruses 13. 3. Ding S, Li Y, Sun Q, Zhu Z, Yu F, Weng S, He J, Dong C.2025. Dualprotection against grouper Rana-Iridovirus (GIV-R) and nervous necrosis virus(NNV) by novel GIV-R(-Δ51)(-)NNV(-CP) chimeric vaccine candidates. FishShellfish Immunol 162:110358. 4. Zhang WF, Duan C, Zhang HT, Weng SP, He JG, Dong CF.2020.Widespread outbreaks of the emerging mandarinfish ranavirus (MRV) both innatural and ISKNV-FKC vaccinated mandarinfish Siniperca chuatsi in Guangdong,South China, 2017. Aquaculture 520. 5. Sun Q, Ding S, Liu X, Zhang W, Yu F, Fu Y, Li Y, Dong C.2025. TheCo-Infection of ISKNV-II and RGNNV Resulting in Mass Mortality of JuvenileAsian Seabass (Lates calcarifer), Zhuhai, Southern China. J Fish Dis 48:e14052. 6. Lin Q, Fu Example 10: Validation of different sample types ISKNV virus solution cultured from MFF-1 cell line was subjected to a 10... 8.2 Infected mandarin fish (approximately 50g each) were collected on day 7 post-infection. Liver, spleen, and kidney tissues were harvested from each infected fish. The tissue was homogenized in 5 mL of PBS containing 0.1% Triton-100 per 1g of tissue, centrifuged, and the supernatant was collected. 60 μL of each sample was used for analysis. Blood samples were diluted 1:1 with PBS containing 0.1% Triton-100, and 60 μL of each sample was used for analysis. Results are shown below. Figure 5 D.

[0071] Example 11: Validation of diluted samples On day 7 post-infection, a homogenate of the spleen tissue from infected fish was used as the stock solution and diluted with PBS in 10-fold serial steps before detection. Both the vp023 and VP101 detection units can detect diluted samples. Figure 5 As shown in C.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus, characterized in that, Two independent chromatography channels are set on the same substrate. Each independent chromatography channel contains a sample pad, a gold label pad, a nitrocellulose membrane containing a detection line T and a control line C, and an absorbent pad, respectively. The sample pad, gold label pad, nitrocellulose membrane containing detection line T and control line C, and absorbent pad are sequentially overlapped on the base plate; the detection line T is located at the end near the conjugate pad, and the control line C is located at the end near the absorbent pad. The gold-labeled pads of the two independent chromatography channels were coated with labeled antibodies for early non-structural proteins and late-stage related proteins of infectious spleen and kidney necrosis virus, respectively. The detection lines T of the two independent chromatography channels are coated with antibodies for detecting early non-structural proteins and late-stage related proteins of infectious spleen and kidney necrosis virus, respectively.

2. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 1, characterized in that, The early non-structural protein includes the early non-structural protein VP023; the late-related protein includes the late-related protein VP101.

3. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 2, characterized in that, The amino acid sequence of the early non-structural protein VP023 is shown in SEQ ID NO: 1; The amino acid sequence of the late-stage related protein VP101 is shown in SEQ ID NO:

2.

4. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 1, characterized in that, The marker antibodies for the early non-structural proteins include the monoclonal antibody ISKNV-3C5; And / or, antibodies for detecting early non-structural proteins include the monoclonal antibody ISKNV-2K10; And / or, marker antibodies for late-stage related proteins include the monoclonal antibody ISKNV-3D15; And / or, antibodies for detecting late-stage related proteins include the monoclonal antibody ISKNV-1B19; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-3C5 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-2K10 is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-3D15 is shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; The amino acid sequence of the heavy chain variable region of the monoclonal antibody ISKNV-1B19 is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

10.

5. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 1, characterized in that, The coating concentration of the detection line T in the two independent chromatography channels is 1-2 mg / mL, preferably 1 mg / mL; The control line C of the two independent chromatography channels is coated with goat anti-mouse IgG at a concentration of 0.5-1 mg / mL, preferably 0.5 mg / mL.

6. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 4, characterized in that, Antibodies labeled with early non-structural proteins or late-related proteins are labeled with colloidal gold particles with a particle size of 20-40 nm.

7. The dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in claim 6, characterized in that, The labeling concentration of the monoclonal antibody ISKNV-3C5 was 17 μg / mL, and the pH of the system during labeling was 8.3-8.5; the labeling concentration of the monoclonal antibody ISKNV-3D15 was 13 μg / mL, and the pH of the system during labeling was 8.8-9.

0.

8. A method for preparing a dual-channel, dual-target test strip for infectious spleen and kidney necrosis virus as described in any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Monoclonal antibodies ISKNV-3C5 and ISKNV-3D15 were conjugated with colloidal gold particles to obtain labeled antibodies for early non-structural proteins and labeled antibodies for late-stage related proteins. (2) The labeled antibodies for early non-structural proteins and late related proteins obtained in step (1) were coated onto the gold pads corresponding to the two chromatography channels, respectively. (3) The detection antibodies for early non-structural proteins and late related proteins are respectively coated onto the nitrocellulose membranes corresponding to the two chromatography channels to form the detection line T for early non-structural proteins and the detection line T for late related proteins, and quality control lines are set. (4) Assemble the sample pad, gold label pad, nitrocellulose membrane and absorbent pad in sequence to obtain the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus.

9. The use of the dual-channel dual-target test strip for infectious spleen and kidney necrosis virus as described in any one of claims 1 to 7 in the preparation of an antigen product for detecting infectious spleen and kidney necrosis virus in fish samples.

10. The application as described in claim 9, characterized in that, The fish samples include at least one of liver, spleen, kidney, and blood samples.

Images