A protein dot blot detection kit for varicella-zoster virus and its application

Abstract

This invention relates to the field of clinical virus detection, and more particularly to a protein dot blot detection kit, detection method, and application for varicella-zoster virus (VZV). The kit includes: a solid-phase carrier membrane for binding and immobilizing proteins in the sample; a blocking agent for blocking non-specific binding sites on the solid-phase carrier membrane; a first antibody that specifically recognizes the VZV antigen; an enzyme-labeled second antibody that binds to the first antibody; and a chromogenic solution for generating a detection signal. This kit is based on protein dot blot technology, detecting proteins with high specificity and a low false-positive rate. The experimental cycle is short, taking only 2-3 hours from sample pretreatment to signal detection, much faster than virus culture (requiring 4-7 days) and PCR detection (although fast, prone to false positives or false negatives), providing rapid evidence for clinical treatment, and is particularly suitable for the diagnosis of acute herpes zoster.

Description

Technical Field

[0001] This invention relates to the field of clinical virus detection, and more particularly to a protein dot blot detection kit for varicella-zoster virus and its application. Background Technology

[0002] Real-time quantitative PCR (qPCR) is currently the mainstream technique for clinical testing of varicella-zoster virus (VZV). However, it still has significant limitations due to factors such as technical principles, sample quality, experimental conditions, and clinical scenarios. For example, stringent sample quality and collection requirements mean that qPCR detection relies on the integrity and concentration of viral nucleic acid in the sample; improper sample collection and processing can directly lead to false negative results. On the one hand, sample types are limited. This technique is effective for detecting samples with high viral loads, such as vesicular fluid and cerebrospinal fluid, but for patients without typical blisters, samples such as skin scrapings and blood have low viral loads, making false negatives likely. On the other hand, improper sample transportation or storage can lead to nucleic acid degradation, and inhibitors in the sample (such as hemoglobin in blood and proteases in tissue samples) can interfere with the PCR amplification reaction, reducing detection sensitivity. Inappropriate qPCR primer design and insufficient probe specificity may lead to non-specific amplification and false positive results; deviations in the concentration ratios of primers, probes, enzymes, and other reagents in the reaction system can also affect detection accuracy. The design of primers and probes, which are typically based on the conserved gene sequences of VZV, is challenging due to limitations in covering viral mutations and rare subtypes. While VZV can mutate or develop rare subtypes during long-term transmission, mutations in the target gene sequence can prevent effective primer or probe binding, leading to false negatives. Furthermore, most clinically used qPCR kits are designed for common VZV strains, offering insufficient coverage for novel variants and requiring regular updates to primer and probe sequences to adapt to viral variations. This technology relies on sophisticated equipment such as qPCR instruments; delays in equipment calibration and errors in fluorescence signal detection can distort quantitative results. Additionally, the high cost of the experiments, including specialized reagents and equipment maintenance, limits its widespread adoption in primary healthcare institutions.

[0003] Therefore, existing technologies need to be improved. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a protein dot blot detection kit for varicella-zoster virus and its application, aiming to solve the problems of strict sample requirements, susceptibility to interference from inhibitors, and possible false negatives / false positives in the clinical application of existing mainstream detection methods (especially real-time quantitative PCR).

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a protein dot blot detection kit for varicella-zoster virus, comprising: Solid-phase support membranes are used to bind and immobilize proteins in the sample to be tested; A blocking agent is used to block non-specific binding sites on the solid support membrane. The first antibody that specifically recognizes the varicella-zoster virus (VZV) antigen; An enzyme-labeled second antibody that binds to the first antibody; The colorimetric solution used to generate the detection signal; The kit is designed to directly spot-detect varicella-zoster virus (VZV) antigen in the test sample via an antigen-antibody reaction.

[0006] Optionally, the solid support membrane is a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose (NC) membrane. Of course, other types of membrane materials can also be selected according to actual experimental needs.

[0007] Optionally, the solid support membrane has a porous structure with a pore size of 0.1 μm to 0.3 μm, preferably 0.2 μm.

[0008] Optionally, the sealing agent is 5% skim milk powder, 1% BSA, or 5% FBS. Other types of sealing agents can also be selected according to actual experimental needs.

[0009] Optionally, the first antibody is an antibody that specifically binds to gE protein, gB protein, gN protein, or ORF9 protein.

[0010] Optionally, the kit further includes a spotting plate disposed on the solid support membrane for spotting samples; The sampling plate is provided with multiple spaced-apart sample holes that run through the sampling plate.

[0011] Optionally, the sample to be tested may be the patient's serum, lysate of peripheral blood mononuclear cells, herpes fluid, cerebrospinal fluid, or tissue homogenate.

[0012] Optionally, the colorimetric solution used to generate the detection signal is a chemiluminescent solution.

[0013] Secondly, the present invention provides the application of the varicella-zoster virus protein dot blot detection kit in the preparation of varicella-zoster virus detection products.

[0014] Optionally, the detection steps include: S1. Obtain the sample to be tested and prepare a sample lysate containing proteins; S2. The sample lysate is directly spotted onto a solid support membrane for binding and fixation; S3. Use a blocking agent to block the non-specific binding sites on the solid support membrane; S4. Incubate with a primary antibody that specifically recognizes the VZV antigen; S5. Incubate with an enzyme-labeled second antibody that binds to the first antibody; S6. Use the colorimetric reagent to generate and detect signals, and determine the presence or load of VZV antigen in the sample based on the presence or intensity of the signal.

[0015] Optionally, the method is used for the clinical diagnosis of acute, subacute, or postherpetic neuralgia phases of herpes zoster and for detecting the effectiveness of antiviral drugs.

[0016] Beneficial Effects: This invention provides a protein dot blot detection kit, detection method, and application for varicella-zoster virus (VZV). The kit allows for direct spot detection of VZV antigen in test samples via antigen-antibody reaction. Based on protein dot blot technology, this kit detects proteins with high specificity and a low false-positive rate. The experimental cycle is short, taking only 2-4 hours from sample pretreatment to signal detection, far faster than virus culture (requiring 4-7 days) and PCR detection (although fast, prone to false positives or false negatives). It can quickly provide a basis for clinical treatment, especially suitable for the diagnosis of acute shingles. Low consumable and reagent costs: No need for electrophoresis gels, transfer buffers, or other Western blotting consumables; reagent usage is low (antibodies, ECL substrates, etc., are used in small quantities). Low instrument requirements: Only a constant temperature shaker and chemiluminescence imaging system are needed; expensive equipment such as PCR instruments and virus incubators are not required. It is easy to configure in clinical laboratories, especially suitable for resource-limited primary healthcare institutions. To compensate for the inefficiency of virus culture: Although virus culture is the "gold standard", it is time-consuming and has a low positive rate (especially when the viral load in clinical samples is low). Dot blot can be used as a rapid screening method, and positive samples can be further cultured for verification to improve diagnostic efficiency.

[0017] In summary, this invention provides a novel VZV detection pathway that differs significantly from existing qPCR technologies in principle and offers complementary advantages. By shifting the detection target from "easily degradable and mutated nucleic acids" to "stable proteins" and the detection platform from "precise nucleic acid amplification instruments" to "flexible immunological detection membranes," it successfully and systematically addresses key clinical pain points in existing technologies, such as high sample requirements, strong equipment dependence, and the risk of false negatives due to mutations. Therefore, it represents a new option with significant clinical application value. Attached Figure Description

[0018] Figure 1This is a schematic diagram of the sample plate structure of the varicella-zoster virus protein dot blot detection kit of the present invention.

[0019] Figure 2 This is a flowchart of the varicella-zoster virus detection method of the present invention.

[0020] Figure 3 This is a flowchart of the varicella-zoster virus detection process for Example 1.

[0021] Figure 4 This is a fluorescence image of ARPE-19 cells infected with fluorescently labeled varicella-zoster virus.

[0022] Figure 5 The image shows the detection results for different protein loading amounts.

[0023] Figure 6 This image shows the results of VZV virus load detection in peripheral blood mononuclear cells of patients at different time periods.

[0024] Figure 7 Images showing the symptoms of the same patient in the acute phase before and after taking the medication.

[0025] Figure 8 The image shows the results of VZV viral load detection in peripheral blood mononuclear cells of patients in the acute phase after taking the medication. Detailed Implementation

[0026] This invention provides a protein dot blot detection kit for varicella-zoster virus and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] "DNA / RNA dot blot" is a rapid qualitative and semi-quantitative detection technique based on the principle of complementary base pairing in nucleic acid molecules. It can directly fix and hybridize DNA or RNA samples for detection without the need for gel electrophoresis separation. It is widely used in gene copy number analysis, viral nucleic acid detection, and preliminary screening of gene expression levels. Therefore, most other viruses reported so far use nucleic acid dot blot, detecting either DNA or RNA. Currently, only PCR is used to detect VZV virus in basic research, detecting it only at the gene level. PCR detects nucleic acids, which are easily degraded and can be degraded by nucleases in the environment and during the operation process, leading to false negatives.

[0028] Currently used antibodies in protein dot blot detection exhibit severe non-specific staining, resulting in a very dark background and a high probability of false positives.

[0029] Based on this, this embodiment provides a protein dot blot detection kit for varicella-zoster virus, comprising: Solid-phase carrier membranes are used to immobilize proteins in test samples. A blocking agent is used to block non-specific binding sites on the solid support membrane. The first antibody that specifically recognizes the VZV antigen; A labeled second antibody that binds to the first antibody; The colorimetric solution used to generate the detection signal; The kit is designed to directly spot-detect VZV antigen in the sample by antigen-antibody reaction.

[0030] It should be noted that this embodiment designs a kit for direct spotting detection of VZV antigens in test samples through antigen-antibody reaction. This kit is based on protein dot blot technology, detecting proteins (i.e., VZV antigens, and simultaneously using four VZV viral antigen proteins, resulting in high specificity; a positive result for all four proteins avoids false positives). The "protein dot blot" in this embodiment detects proteins; VZV virus is a cellular virus, meaning it cannot be detected through serum or other body fluids, as it prefers to hide inside cells. Using this kit, peripheral blood mononuclear cells from patients can be isolated using peripheral blood separation reagents and cell lysis buffer (both commercially available). After lysis, the virus hidden within the peripheral blood mononuclear cells can be detected. The experimental cycle is short, from sample pretreatment to signal detection, taking only 2-3 hours, much faster than virus culture (requiring 4-7 days) and PCR detection (although fast, prone to false positives or false negatives), providing rapid evidence for clinical treatment, especially suitable for the diagnosis of acute herpes zoster. Based on antigen-antibody specific binding, the false positive rate is low. Consumables and reagent costs are low: no electrophoresis gels, transfer buffers, or other Western blotting consumables are required, and reagent usage is minimal (antibodies, ECL substrates, etc., are used in small quantities). Instrument requirements are low, requiring no high-end equipment: only a constant-temperature shaker and chemiluminescence imaging system are needed; expensive equipment such as PCR instruments and virus incubators are not required, making it easy to configure in clinical laboratories, especially suitable for resource-constrained primary healthcare institutions. It compensates for the inefficiency of virus culture: although virus culture is the "gold standard," it is time-consuming and has a low positive rate (especially when viral load is low in clinical samples). Dot blot can serve as a rapid screening method, and positive samples can be further cultured for verification, improving diagnostic efficiency.

[0031] In some embodiments, the solid support membrane is a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose (NC) membrane.

[0032] This embodiment can also select other types of membrane materials according to actual experimental needs. A single solid-phase carrier membrane can detect multiple samples simultaneously: for example, a PVDF membrane can be designed with 30-60 sampling sites, which can simultaneously detect batches of clinical samples (such as epidemiological screening, preoperative screening of hospitalized patients), greatly improving detection efficiency and reducing the unit sample detection cost.

[0033] In some embodiments, the pore size of the solid support membrane is from 0.1 μm to 0.3 μm, and can be 0.1 μm, 0.2 μm, or 0.3 μm, preferably 0.2 μm.

[0034] It should be noted that the solid-phase support membrane (such as a PVDF membrane) is specifically designed for this kit. Using such a membrane increases the effective binding sites per unit area, thereby enhancing protein binding. The binding capacity is negatively correlated with pore size (the smaller the pore size, the more effective binding sites per unit area). The protein binding capacity of a 0.2 μm pore size PVDF membrane is typically 200-300 μg / cm³. 2 (High binding capacity), PVDF membranes with pore sizes greater than 0.45 μm (100-150 μg / cm³) 2 Or a 0.22μm pore size PVDF membrane (150-250μg / cm³) 2 Furthermore, the smaller pore size can limit the lateral diffusion of target molecules within the membrane, resulting in narrower bands and clearer edges (low diffusivity).

[0035] In some embodiments, the sealing agent is 5% skim milk powder (e.g., Beyotime Pharmaceuticals, P0216).

[0036] In some embodiments, the first antibody is an antibody that specifically binds to gE protein, gB protein, gN protein, or ORF9 protein.

[0037] It should be noted that the gE protein antibody was selected from AB52550 from Abcam; the gB protein antibody was selected from AB272686 from Abcam; the gN protein antibody was selected from ZP001 from Shenzhen Zhipu Biotechnology Co., Ltd.; and the ORF9 protein antibody was selected from P002 from Shenzhen Zhipu Biotechnology Co., Ltd. The first antibody in this embodiment has high specificity, avoiding false positives. This embodiment uses antibodies targeting the virus's own antigens. Utilizing the specific reaction between the antigen and antibody, VZV can be effectively distinguished from other herpesviruses (such as HSV-1 / 2). Currently, no antibody for dot blot has been reported; only antibodies suitable for ELISA and Western blotting exist. However, antibodies used in ELISA and Western blotting exhibit severe non-specific staining, resulting in a very dark background and poor measurement results. This embodiment can not only determine the presence of VZV antigen in the sample (qualitative diagnosis of infection) but also quantify the spot signal intensity using ImageJ software, semi-quantitatively assessing viral load and providing a reference for clinical assessment of the degree of infection.

[0038] In some embodiments, the kit further includes a spotting plate disposed on the solid support membrane for spotting samples; The sampling plate is provided with multiple spaced-apart sample holes that extend through the plate, such as... Figure 1 As shown in the figure, the holes are for adding samples.

[0039] It should be noted that this embodiment features spaced-apart sample wells on the sample dispensing plate. These wells penetrate the plate and correspond to the solid support membrane, ensuring a preset distance between adjacent wells. This allows each sample to form an independent diffusion region, effectively preventing physical mixing and cross-contamination between samples and ensuring the accuracy of experimental results. Alternatively, an array of wells can be used, achieving consistent sampling distances across multiple wells. This helps image analysis software accurately identify and locate each sample point, improving the accuracy and efficiency of data analysis and reducing manual calibration.

[0040] In some embodiments, the sample to be tested is the patient's serum, lysate of peripheral blood mononuclear cells, herpes fluid, cerebrospinal fluid, or tissue homogenate.

[0041] It should be noted that the sample processing in this embodiment is simple and requires no complex pretreatment: clinical samples (serum, herpes fluid, cerebrospinal fluid, tissue homogenate) only need to be centrifuged to remove impurities, without the need for SDS-PAGE electrophoresis and membrane transfer steps, and can be directly spotted for detection, avoiding the influence of sample protein degradation and denaturation on the results (especially suitable for easily inactivated samples such as herpes fluid). It is compatible with micro-samples: some clinical samples (such as cerebrospinal fluid, micro-volume herpes fluid) have limited volume or low protein concentration. Dot blot only requires a small amount of sample (e.g., 5 μl) for detection, and has low requirements for protein integrity, making it more suitable for micro-clinical samples than Western blotting.

[0042] In some embodiments, the colorimetric solution used to generate the detection signal is a chemiluminescent solution (Beyotime Biotechnology Co., Ltd., P0018M).

[0043] This embodiment also provides an application of a protein dot blot detection kit for varicella-zoster virus in the preparation of varicella-zoster virus detection products.

[0044] like Figure 2 As shown, the detection steps include: S1. Obtain the sample to be tested and prepare a sample lysate containing proteins; S2. The sample lysate is directly spotted onto a solid support membrane for binding and fixation; S3. Use a blocking agent to block the non-specific binding sites on the solid support membrane; S4. Incubate with a primary antibody that specifically recognizes the VZV antigen; S5. Incubate with an enzyme-labeled second antibody that binds to the first antibody; S6. Use the colorimetric reagent to generate and detect signals, and determine the presence or load of VZV antigen in the sample based on the presence or intensity of the signal.

[0045] It should be noted that the method described in this embodiment has a short cycle time, taking only 2-3 hours from sample preprocessing to signal detection, which is much faster than virus culture (which requires 4-7 days) and PCR detection (although fast, it is prone to false positives or false negatives). It can quickly provide a basis for clinical treatment, and is especially suitable for the diagnosis of acute herpes zoster. The operation procedure is simplified and does not require professional electrophoresis / transfer skills: clinical laboratory technicians can get started with simple training, without relying on complex equipment for Western blotting (such as electrophoresis apparatus and transfer apparatus), lowering the operation threshold and making it suitable for rapid testing in primary hospitals or emergency departments.

[0046] In one embodiment, the method is used for the clinical diagnosis of acute, subacute, or postherpetic neuralgia phases of herpes zoster and for detecting the effectiveness of antiviral drugs.

[0047] It should be noted that the clinical course of herpes zoster exhibits distinct phases. Based on the duration of the illness, herpes zoster can be divided into the acute phase (AHN), subacute phase (SHN) (duration: 1-3 months), and postherpetic neuralgia (PHN) (duration > 3 months). Currently, it has been reported that the virus may be present in each phase, but not every patient has it in each phase. Therefore, detecting the presence of the virus is crucial for clinical drug treatment.

[0048] This embodiment provides the use of protein dot blot technology in the preparation of kits for diagnosing or monitoring varicella-zoster virus (VZV) infection.

[0049] The present invention will be further described below through specific embodiments.

[0050] Example 1 This embodiment uses a constructed cell model infected with varicella-zoster virus, including the following steps: ARPE-19 cells (human retinal pigment epithelial cells) were routinely cultured, then infected with VZV virus for 24 hours, and simultaneously labeled with VZV-specific fluorescent markers. The results are as follows. Figure 4 As shown, this indicates successful viral infection. Sample preparation: ARPE-19 cells infected with VZV were collected, lysed with lysis buffer, and total protein was extracted. Uninfected VZV cell lysate was used as a control. Dot blot detection: Samples (uninfected / infected) with different protein loading amounts (1.25 μg, 2.5 μg, 5 μg, 10 μg) were spotted onto a solid membrane. After blocking, incubation with primary antibodies against VZV-specific proteins (ORF9, gN, gE, gB), and binding with secondary antibodies, the spot signals were detected by fluorescence imaging. Figure 5 As shown. From Figure 4 , Figure 5 It was found that by infecting ARPE-19 cells with VZV virus to establish a cell infection model, and by detecting different protein loading amounts using protein dot blot technology, the signals of the four VZV specific proteins—ORF9, gN, gE, and gB—gradually increased with increasing protein loading amounts. However, no signal values ​​were detected in the lysates of uninfected cells. These results demonstrate that protein dot blot detection of intracellular VZV virus has high sensitivity and specificity.

[0051] Example 2 A method for detecting varicella-zoster virus, such as Figure 3As shown, it includes the following steps: Step 1, Sample collection: Collect whole blood from shingles patients (only 2 ml is needed).

[0052] Step 2, Isolation of peripheral blood mononuclear cells: Peripheral blood mononuclear cells are isolated from the patient's whole blood using a peripheral blood mononuclear cell isolation reagent.

[0053] Step 3, Sample Preparation: Lyse peripheral blood mononuclear cells on ice using cell lysis buffer, then centrifuge at 13,000 rpm for 5 minutes. Transfer to a new EP tube. Remaining samples can be stored at -80°C for analysis.

[0054] Step 4, Spotting: Spot the prepared sample onto the solid support membrane of the kit using Dot blot experimental technique (with spotting device assistance).

[0055] Step 5: Drying to increase protein binding and fixation: After spotting, place at 42℃ for 5 minutes. The purpose is to allow the sample to dry thoroughly and adsorb onto the solid support membrane.

[0056] Step 6, Blocking, Primary Antibody and Secondary Antibody Incubation: After spotting, block with blocking solution. Then incubate with primary and secondary antibodies (anti-gE, anti-gB, anti-gN, anti-ORF9).

[0057] Step 7, Signal Detection: After antibody incubation, add chemiluminescent solution and detect the signal using a chemiluminescence analyzer.

[0058] Step 8, Observation and Evaluation: The ImageJ software was used to quantify the spot signal intensity and semi-quantitatively evaluate the viral load.

[0059] The difference between this embodiment and Embodiment 1 is that in this embodiment, peripheral blood mononuclear cells from 5 healthy individuals, 14 patients with acute herpes zoster (AHN-14), 7 patients with subacute herpes zoster (SHN-7), and 4 patients with postherpetic neuralgia (PHN-4) were used for comparison. Peripheral blood mononuclear cells were isolated using a peripheral blood separation kit. Cells were lysed with lysis buffer, and total protein was extracted and detected by Dot blot. Proteins from peripheral blood mononuclear cells from different patients at different time points were spotted onto a solid membrane, sequentially blocked, incubated with primary antibodies against VZV-specific proteins (ORF9, gN, gE, gB), and then bound with labeled secondary antibodies. The spot signals were detected by fluorescence imaging, and the results are shown below. Figure 6 As shown. Figure 6 This image shows the viral load of VZV in peripheral blood mononuclear cells of patients with herpes zoster at different stages. Figure 6 In Figure A, the detection of VZV load in peripheral blood mononuclear cells of healthy individuals is shown. Figure 6In section B, the detection of VZV load in peripheral blood mononuclear cells of patients in the acute herpetic neuralgia (AHN) phase was performed. Figure 6 In the middle section, C represents the detection of VZV load in peripheral blood mononuclear cells of patients in the subacute herpetic neuralgia (SHN) phase. Figure 6 The D-value represents the detection of VZV load in peripheral blood mononuclear cells from patients with postherpetic neuralgia (PHN). Figure 6 It can be seen that protein dot blot can detect different viral loads of VZV in peripheral blood mononuclear cells of different patients with herpes zoster at different clinical stages.

[0060] Example 3 The difference between this embodiment and Embodiment 1 is that the peripheral blood mononuclear cells used in this embodiment are from the first day of admission of an acute herpetic neuralgia (AHN) patient, and the patient was given acyclovir for 4 days as prescribed (herpes zoster significantly subsided, scabbed over, and had no obvious symptoms). The symptoms were as follows. Figure 7 As shown. By separating as Figure 7 The peripheral blood mononuclear cells were lysed after four days of acyclovir administration as prescribed. Total protein was extracted and analyzed by dot blot. The obtained protein samples from the peripheral blood mononuclear cells were spotted onto a solid membrane, sequentially blocked, incubated with primary antibodies against VZV-specific proteins (ORF9, gN, gE, gB), and then bound with labeled secondary antibodies. The spot signals were detected by fluorescence imaging. The results are shown below. Figure 8 As shown. From Figure 8 It is known that the protein dot blot of this embodiment can track and detect the viral load of VZV in peripheral blood mononuclear cells of the same patient before and after drug administration, and can thus indirectly reflect the effect of antiviral drugs.

[0061] In summary, this invention provides a protein dot blot detection kit, detection method, and application for varicella-zoster virus (VZV). This invention designs a kit that can directly detect VZV antigens in test samples through antigen-antibody reactions. This kit is based on protein dot blot technology, detecting proteins (i.e., VZV antigens, and simultaneously using four VZV viral antigen proteins, resulting in high specificity and avoiding false positives). In this embodiment, the "protein dot blot" detects proteins; VZV virus is a cellular virus, meaning it cannot be detected through serum or other body fluids, as it prefers to hide within cells. Using this kit, cell lysis buffer (commercially available) can be used to isolate peripheral blood mononuclear cells from patients, allowing detection of the virus within these cells. The experimental cycle is short, with the entire process from sample pretreatment to signal detection taking only 2-3 hours, far faster than virus culture (requiring 4-7 days) and PCR detection (although fast, prone to false positives or false negatives), providing rapid evidence for clinical treatment, especially suitable for the diagnosis of acute shingles. Based on antigen-antibody specific binding, the false positive rate is low. Consumables and reagent costs are low: no electrophoresis gels, transfer buffers, or other Western blotting consumables are required, and reagent usage is small (antibodies, EC substrates, etc., are used in small quantities). Instrument requirements are low, requiring no high-end equipment: only a constant temperature shaker and chemiluminescence imaging system are needed; expensive equipment such as PCR instruments and virus incubators are not required, making it easy to configure in clinical laboratories, especially suitable for resource-constrained primary healthcare institutions. It compensates for the inefficiency of virus culture: although virus culture is the "gold standard," it is time-consuming and has a low positive rate (especially when the viral load in clinical samples is low). Dot blot can be used as a rapid screening method, and positive samples can be further cultured for verification, improving diagnostic efficiency.

[0062] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A protein dot blot detection kit for varicella-zoster virus, characterized in that, include: Solid-phase support membranes are used to bind and immobilize proteins in the sample to be tested; A blocking agent is used to block non-specific binding sites on the solid support membrane. The first antibody that specifically recognizes the varicella-zoster virus antigen; An enzyme-labeled second antibody that binds to the first antibody; The colorimetric solution used to generate the detection signal; The kit is designed to directly spot-detect varicella-zoster virus antigen in test samples via antigen-antibody reaction.

2. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The solid support membrane is a polyvinylidene fluoride membrane or a cellulose nitrate membrane.

3. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The solid support membrane has a porous structure with a pore size of 0.1 μm to 0.3 μm.

4. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The first antibody is an antibody that specifically binds to gE protein, gB protein, gN protein, or ORF9 protein.

5. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The kit also includes a spotting plate, which is disposed on the solid support membrane for spotting samples; The sampling plate is provided with multiple spaced-apart sample holes that run through the sampling plate.

6. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The samples to be tested are the patient's serum, lysate of peripheral blood mononuclear cells, herpes fluid, cerebrospinal fluid, or tissue homogenate.

7. The varicella-zoster virus protein dot blot detection kit according to claim 1, characterized in that, The colorimetric solution used to generate the detection signal is a chemiluminescent solution.

8. The use of a protein dot blot assay kit for varicella-zoster virus according to any one of claims 1-7 in the preparation of varicella-zoster virus detection products.

9. The application according to claim 8, characterized in that, The testing steps include: S1. Obtain the sample to be tested and prepare a sample lysate containing proteins; S2. The sample lysate is directly spotted onto a solid support membrane for binding and fixation; S3. Use a blocking agent to block the non-specific binding sites on the solid support membrane; S4. Incubate with a primary antibody that specifically recognizes the VZV antigen; S5. Incubate with an enzyme-labeled second antibody that binds to the first antibody; S6. Use the colorimetric reagent to generate and detect signals, and determine the presence or load of VZV antigen in the sample based on the presence or intensity of the signal.

10. The application according to claim 9, characterized in that, The detection steps are used for the clinical diagnosis of acute, subacute, or postherpetic neuralgia phases of herpes zoster, as well as to detect the effectiveness of antiviral drugs.

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