ELISpot technology-based method for detecting infectivity of oncolytic virus in urine and swab preserving fluid

Abstract

The invention discloses a method for detecting the infectivity of oncolytic viruses in urine and swab preserving fluid based on an ELISpot technology, and relates to the technical field of detection of the infectivity of the oncolytic viruses in the urine and swab preserving fluid, and the specific method comprises the following steps: firstly, placing a proper amount of cells in a transparent cell culture plate for overnight culture, then transferring a sample into a cell plate, and carrying out ELISA detection on the cell plate; culturing for a period of time. An indirect immunofluorescence method is established by adopting an immunofluorescence technology and taking a rabbit anti-Vaccinia Virus Polyclonal Antibody polyclonal antibody as a primary antibody and an Alexa FluorR 488 labeled goat anti-rabbit antibody as a secondary antibody, and the virus infectivity is judged according to the quantity of fluorescent cells infected by the virus. The invention fills the technical blank in the field of detecting the infectivity of the oncolytic virus by using the ELISpot technology, is accurate in detection and high in sensitivity, and has important significance for new drug research and development of oncolytic virus drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of oncolytic virus infectivity detection in urine and swab preservation fluid, and in particular to a method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology. Background Art

[0002] Oncolytic viruses are a new and unique cancer immunotherapeutic agent that can selectively eliminate tumor cells by targeting cytotoxicity and simultaneously inducing systemic anti-tumor immune responses, while leaving normal cells unharmed. Currently, four oncolytic viruses have been approved worldwide for the treatment of cancer, namely RIGVIR in Latvia in 2004, Oncorine in China in 2005, T-VEC in the United States in 2015, and Delytact in Japan in 2021. Oncolytic viruses are a highly versatile cancer treatment platform that has the ability to selectively replicate in tumor cells and induce tumor cell lysis, and can induce immune cell infiltration at the tumor site through a virus-mediated inflammatory response, turning a "cold tumor" into a "hot tumor." Oncolytic viruses induce immunogenic cell death, and the subsequent release of tumor-associated neoantigens and damage-associated molecular patterns can effectively activate dendritic cell maturation, recruit and activate immune cells such as T cells to infiltrate into the immune-rejecting or immunosuppressive tumor microenvironment, and improve the recognition and killing ability of immune cells, thereby driving anti-tumor immunity and alleviating the immunosuppressive tumor microenvironment, thereby triggering a potent anti-tumor immune response. Local injection of oncolytic viruses into a single tumor site can induce abscopal effects, promote innate and adaptive antitumor immune responses, and distant uninfected tumors will also experience immune-mediated rejection reactions leading to tumor regression. The effectiveness of oncolytic viruses against tumors mainly depends on their powerful ability to induce antigen-specific antitumor immune responses. In addition, oncolytic viruses can be genetically engineered to express therapeutic genes that can further enhance antitumor activity. Oncolytic viruses can also help regulate abnormalities in the tumor microenvironment, such as neovascularization, tumor metabolism, and the rigid extracellular matrix barrier brought by tumor stromal cells.

[0003] Viral shedding of oncolytic virus drugs refers to the spread of oncolytic viruses through the patient's secretions / excretions. Viral shedding focuses on the assessment of the virus to third parties or possible environmental risks. Examining viral shedding helps guide the monitoring of long-term adverse reactions in clinical studies. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) has developed a consideration document on viral shedding and vector shedding, which details the detection methods for viral shedding and considerations in the design of non-clinical research trials. Therefore, the risk of viral shedding needs to be assessed in clinical trials. Summary of the invention

[0004] In view of the technical gap in the field of detection methods for detecting oncolytic virus infectivity in urine and swab preservation fluid using ELISpot technology, the present invention provides a detection method for oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology, firstly placing an appropriate amount of Vero cells in a transparent cell culture plate for overnight culture, then transferring the sample to a cell plate, and culturing for a period of time. Immunofluorescence technology is used, rabbit anti-Vaccinia Virus Polyclonal Antibody polyclonal antibody is used as the primary antibody, Alexa FluorR 488 labeled goat anti-rabbit antibody is used as the secondary antibody, and an indirect immunofluorescence method is established, and the amount of fluorescent cells infected by the virus is detected by the ELISpot instrument to determine the virus infectivity.

[0005] The above-mentioned purpose of the present invention is specifically achieved through the following technologies:

[0006] The method for detecting the infectivity of oncolytic viruses in urine and swab preservation fluid based on ELISpot technology includes the following steps:

[0007] S1. Inoculate Vero cells into a 96-well cell plate and culture in an incubator overnight;

[0008] S2, diluting the sample using cell growth medium;

[0009] S3. When the confluence of Vero cells is ≥80%, the culture medium in the 96-well plate is discarded, and the sample diluted in step S2 is added and cultured in a cell culture incubator;

[0010] S4, discard the culture medium in the Vero cell 96-well plate, dry at room temperature for 5 minutes; add immunostaining fixative along the wall and incubate, then remove the fixative and wash the cells in the wells with washing solution;

[0011] S5, cells were added to the 96-well plate and incubated at room temperature in the dilution buffer, and then washed with washing solution;

[0012] S6, add primary antibody working solution to the cell 96-well plate, and wash with washing solution after incubation;

[0013] S7. Add the secondary antibody working solution to the 96-well cell plate, wash with washing solution after incubation; then use the ELISPOT instrument for detection.

[0014] Preferably, in step S1, 100 μL of Vero cell suspension is added to the 96-well cell plate, and the concentration of the Vero cell suspension is 2.00×10 5 cells / mL, Vero cell number 2.00×10 4 indivual.

[0015] Preferably, in step S1, the culture conditions are 5% CO2 and the culture temperature is 37°C.

[0016] Preferably, in step S2, the sample is urine or swab preservation fluid.

[0017] Preferably, in step S2, the cell growth medium is a complete cell culture medium, and 50 mL of fetal bovine serum and 5 mL of streptomycin-penicillin solution are added to 450 mL of 1640 culture medium.

[0018] Preferably, in step S2, the sample is diluted to twice the volume using cell growth medium.

[0019] Preferably, in step S3, the amount of diluted sample added is 100 μL / well, the culture temperature is 37° C., and the culture time is 24 h.

[0020] Preferably, in the steps S4-S7, the washing solution used in the washing process is 200 μL 1×PBS buffer, pH=7.4.

[0021] Preferably, in step S4, the fixative used is immunostaining fixative (Biyuntian, P0098-500mL), and the amount added is 100 μL / well.

[0022] Preferably, in step S4, the incubation temperature is room temperature, the incubation time is 15 minutes, and the washing times are 2 times.

[0023] Preferably, in step S5, the dilution buffer used is a 1×PBS solution containing 1 wt.% BSA, 0.05% Proclin-300 by volume and 0.05% Tween-20 by volume, with a pH of 7.4.

[0024] Preferably, in step S5, the amount of dilution buffer added is 200 μL / well, the incubation time is ≥1 h, and the number of washes is 3 times.

[0025] Preferably, in step S6, the primary antibody working solution used is prepared by using Vaccinia Virus Polyclonal Antibody and dilution buffer at a volume ratio of 1:400.

[0026] Preferably, in step S6, the amount of primary antibody working solution added is 25 μL / well, the incubation temperature is 37° C., the incubation time is 1 h±5 min, and the number of washes is 3 times.

[0027] Preferably, in step S7, the secondary antibody working solution used is Goat Anti-Rabbit IgG H&L (Alexa Fluor R 488) prepared with dilution buffer at a volume ratio of 1:200.

[0028] Preferably, in step S7, the amount of the secondary antibody working solution added is 25 μL / well, the incubation temperature is 37° C., the incubation time is 1 h±5 min, and the number of washes is 3 times.

[0029] Preferably, in step S7, the detection parameters of the ELISpot cytometer are as shown in Table 1 below.

[0030] Table 1 ELISpot cytometer detection parameters

[0031]

[0032] More preferably, the step S1 also includes the recovery and subculture process of Vero cells.

[0033] More preferably, the specific steps of the Vero cell recovery process are: first take the frozen cells and place them in a 37°C water bath until the cells are thawed; then add the thawed cell suspension into the cell growth medium and blow and mix, and discard the supernatant after centrifugation; then add the cell growth medium to resuspend and mix, and incubate at a constant temperature of 37°C in a 5% CO2 environment to complete the cell recovery.

[0034] More preferably, the specific steps of the Vero cell passaging process are: first take the revived cells, rinse and discard the old culture medium; then add 0.25wt.% trypsin solution, incubate at 37°C in a 5% CO2 environment until the cells are observed to be completely detached from the bottom of the culture container by the naked eye; then add cell growth culture medium to resuspend and blow, count the cells, and prepare a cell passaging solution.

[0035] Compared with the prior art, the advantages of the present invention are as follows: the present invention uses ELISpot technology for the first time to directly detect the infectivity of oncolytic viruses, filling the gap in the field and providing accurate detection, which is of great significance for the development of new oncolytic virus drugs; the entire analysis process is simple to operate and has good repeatability; it has high sensitivity, with a detection limit of 5.00E+03pfu / mL for human urine and 2.50E+03pfu / mL for swab preservation fluid. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a high-concentration positive quality control effect display diagram in the embodiment, and the left and right figures represent repeated experiments;

[0037] Figure 2This is a diagram showing the effect of low-concentration positive quality control in the example. The left and right diagrams represent repeated experiments.

[0038] Figure 3 This is a display diagram of the negative quality control effect in the embodiment, and the left and right figures represent repeated experiments. DETAILED DESCRIPTION

[0039] The technical scheme of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of embodiments of the present invention, rather than all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work conditions belong to the scope of protection of the present invention. If there are no special instructions, the experimental operations in the embodiments all adopt the mature prior art in the biological field or the chemical field, and the raw materials and reagents used can be obtained by commercial purchase.

[0040] Example

[0041] 1. Experimental drugs

[0042] (1) Vero cells: Cell2022009, specification 2×10 7 pcs / 5mL, China Center for Type Culture Collection, stored in liquid nitrogen;

[0043] (2) Positive control: oncolytic vaccinia virus, stored at -90°C to -60°C, supplied by Heyuan Biotechnology (Shanghai) Co., Ltd., specification / concentration 5.25E+08 pfu / mL;

[0044] (3) Primary antibody: Vaccinia Virus Polyclonal Antibody, supplier: Thermo, catalog number PA17258, batch number, XL3774473, specification concentration 4 mg / mL-5 mg / mL;

[0045] (4) Secondary antibody: Goat Anti-Rabbit IgG H&L (Alexa Fluor R 488), supplier Abcam, catalog number Ab150077, batch number GR3445008-1, specification / concentration 2 mg / mL;

[0046] (5) Fetal bovine serum (FBS): supplier Gibco, catalog number 10099-141;

[0047] (6) Blank matrix: individual human urine, all negative urine not infected by the virus. Storage condition ≤ -10℃.

[0048] 2. Test consumables and instruments

[0049] The test instruments and consumables are shown in Table 2 below.

[0050] Table 2 List of test instruments or consumables

[0051]

[0052]

[0053]

[0054] 3. Preparation of test solution

[0055] (1) Cell growth medium (CGM): Aseptically measure 50 mL of fetal bovine serum (FBS) and 5 mL of penicillin-streptomycin (Pen Strep), add to 445 mL of high-glucose DMEM medium, mix well and set aside; store at 2-8°C, with a shelf life of 3 months.

[0056] (2) Dilution buffer (DBS, 1×PBS containing 1% BSA, 0.05% Proclin-300 and 0.05% Tween-20): Measure 10 mL of 20×PBS and add it to 190 mL of ultrapure water to obtain 1×PBS. Add 2 g BSA, 100 μL Proclin-300 and 100 μL Tween-20, mix well and set aside; store at 2-8°C, with a shelf life of 1 month.

[0057] (3) Primary antibody working solution: Prepare the primary antibody working solution by diluting Vaccinia Virus Polyclonal Antibody (Stock 2) with dilution buffer at a ratio of 1:400, mix well and set aside; store at room temperature and use immediately after preparation.

[0058] (4) Fluorescent secondary antibody working solution: Prepare fluorescent secondary antibody working solution by mixing Goat Anti-Rabbit IgG H&L (Alexa Fluor R 488) (Stock 3) with dilution buffer at a ratio of 1:200, mix well and set aside; store at room temperature and use immediately after preparation.

[0059] (5) Negative control sample (NC): NC is CGM, used as a negative control sample in this experiment.

[0060] (6) Positive quality control samples: The positive control was spiked into NC to prepare a series of positive samples with different concentrations.

[0061] Oncolytic vaccinia virus (Stock 1) with a concentration of 5.25E+08 pfu / mL was used for positive sample preparation, and the formula is shown in Table 3 below.

[0062] Table 3 Positive sample preparation method

[0063]

[0064] 4. Analysis methods

[0065] (1) Cell recovery

[0066] ① Turn on the constant temperature water bath and preheat to 38℃;

[0067] ② Add about 9 mL of cell growth medium to a 15 mL centrifuge tube;

[0068] ③ Take out a frozen cell from the Vero working cell bank frozen in the liquid nitrogen tank, immediately immerse the body of the cryopreservation tube in a water bath and gently shake it to melt the frozen cells until only a small ice crystal remains in the middle, take out the cryopreservation tube, disinfect it with alcohol and place it in a sterile operating table;

[0069] ④ Transfer all the suspension in the cryopreservation tube to the centrifuge tube in step 2 and mix well by pipetting;

[0070] ⑤ Centrifuge at 200g for 5 minutes in a refrigerated centrifuge and discard the supernatant;

[0071] ⑥ Add about 8 mL of cell growth medium to resuspend and mix, transfer to a T25 culture flask, shake gently, and culture in a carbon dioxide constant temperature incubator at 37°C, 5% CO2. Pay attention to check the cell status.

[0072] (2) Cell passaging

[0073] The passage time (Table 4) and passage-related parameters (Table 5) in this method are given only based on the previous cell culture data. The cells can be passaged when the cell confluence reaches about 80% according to the actual situation, and the passage strategy can be adjusted based on this. Each time the cells are dissociated with 0.25% Trypsin, the cell passage number is +1.

[0074] ①Take out the cells from the carbon dioxide incubator and place them in a medical clean bench;

[0075] ② Aspirate and discard the old culture medium, gently rinse the cells with an appropriate amount of PBS pH7.4 (1×) basal culture medium, and aspirate and discard 1 mL of PBS pH7.4 (1×);

[0076] ③ Add an appropriate amount of 0.25% Trypsin solution (see Table 5), and incubate the culture flask at 37°C, 5% CO2 for 5 minutes. After the incubation, the cells can be observed to be detached from the bottom of the culture flask. If they have not detached, continue incubation until the cells detach;

[0077] ④ Add an appropriate amount of cell growth medium (see Table 5) to the culture flask, mix by pipetting, transfer all the suspension to a centrifuge tube, centrifuge at 200g for 5 minutes, and discard the supernatant. Add an appropriate amount of cell growth medium (see Table 5), mix by pipetting, and take two 20μL portions in parallel for cell counting;

[0078] ⑤ Prepare the cell passage solution according to the results of cell counting, and calculate the volume of cell suspension to be used according to the following formula:

[0079] Cell suspension volume (mL) = total number of cells required (cells) / viable cell density obtained by counting (cells / mL);

[0080] ⑥ Add the cell suspension according to the volume obtained in step ⑤ into the corresponding culture bottle pre-stored with an appropriate amount of cell growth medium (see Table 5), shake well and culture in an incubator at 37°C, 5% CO2.

[0081] Table 4 Cell subculture cycle

[0082]

[0083]

[0084] Table 5 Cell passaging related parameters

[0085] Culture bottle model T25 T75 T150 PBS volume for rinsing (mL) 5 10 10 Trypsin volume for digestion (mL) 1 2 4 CGM volume for digestion termination (mL) 4 8 16 Pre-stored CGM volume in culture bottle (mL) 8 15 30

[0086] (3) Cell culture medium replacement

[0087] If the cells have not reached the required confluence for passaging and the medium has turned yellow, or the medium needs to be changed for other reasons, it can be replaced according to the following steps.

[0088] ① Aspirate and discard the old culture medium, rinse the cells gently with about 10 mL of PBS pH 7.4 (1×) basal culture medium, and aspirate and discard the PBS pH 7.4 (1×) basal culture medium;

[0089] ② Add cell growth medium of equal volume to the old medium as needed, shake well and culture in an incubator at 37°C, 5% CO2.

[0090] (4) Cell counting

[0091] The cell number should be calculated when subculturing and plating. The calculation method is as follows:

[0092] ① Add 20 μL of trypan blue (0.4%) solution to the 20 μL cell suspension sampled in parallel, and mix by pipetting;

[0093] ② Take 10 μL of each of the two parallel samples and load them onto the machine. Use a cell counter with a VERO template (see Table 6) to determine the density of live cells. The %CV of the two measurements should not be greater than 15.0%;

[0094] ③ The average of the two measurements of live cell density was used as the reported live cell density, and the average of the two measurements of cell viability was used as the reported cell viability. The average live cell density used for plating should not be less than 90%.

[0095] Table 6 Vero cell counter parameters

[0096]

[0097] (4) Virus shedding detection:

[0098] ① Cultivate to 2.00×10 5 Vero cells at 100 μL / mL were inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 5% CO2, 37°C incubator;

[0099] ② Dilute the high-concentration positive quality control sample, low-concentration positive quality control sample and negative quality control sample to twice their original concentration using cell growth medium;

[0100] ③When the confluence of Vero cells is ≥80%, discard the medium in the 96-well plate and add 100 μL / well of the diluted sample in ② and culture in a cell culture incubator at 37°C for 24 h;

[0101] ④ Aspirate the culture medium in the Vero cell 96-well plate and dry it at room temperature for 5 minutes; add immunostaining fixative along the wall at 100 μL / well and incubate at room temperature for 15 minutes. After incubation, aspirate the fixative and wash the cells in the plate twice with 200 μL / well of washing solution;

[0102] ⑤ Add dilution buffer to the cells in a 96-well plate at 200 μL / well and incubate at room temperature for 1 hour, then wash three times with washing solution;

[0103] ⑥ Add primary antibody working solution to the cell 96-well plate at 25 μL / well, incubate at 37°C for 1h±5min, and then wash 3 times with washing solution;

[0104] ⑦ Add the secondary antibody working solution to the cell 96-well plate at 25 μL / well, incubate at 37°C for 1h±5min, and then wash 3 times with washing solution; use ELISPOT instrument for detection. The detection parameters of ELISpot cytometer are shown in Table 1.

[0105] The dilution buffer used was a 1×PBS solution containing 1wt.% BSA, 0.05% Proclin-300 and 0.05% Tween-20, pH=7.4; the washing solution was a 1×PBS buffer, pH=7.4. The high concentration positive quality control chart, the low concentration positive quality control chart, and the negative quality control chart are shown in Figure 1. Figure 1-3 shown.

[0106] (5) Detection limit

[0107] The detection limit of the method refers to the minimum concentration of the virus in the matrix that can detect a positive reaction. The test drug was prepared into a LOD sample (5.00E+06pfu / mL, the matrix content must be above 95%) with urine and swab preservation solution (non-inactivated), and stored in a -80℃ refrigerator for at least 12 hours. On the day of analysis, the sample was filtered with a 0.45μm sterile filter and diluted with cell growth medium. According to the results of positive and negative quality control samples ( Figure 1-3 ), and the number of detections at each level of the LOD serial dilution samples (fluorescence plate points ≥ 20) was summarized, and the lowest concentration level with a detection rate of more than 95% was defined as the detection limit of the method; the test drug was oncolytic bean seedling virus.

[0108] Use the following method:

[0109] ① Cultivate to 2.00×10 5 Vero cells at 100 μL / mL were inoculated into a 96-well plate, 100 μL per well, and cultured overnight in a 5% CO2, 37°C incubator;

[0110] ② Dilute the LOD series samples to twice their original concentration using cell growth medium;

[0111] ③When the confluence of Vero cells is ≥80%, discard the medium in the 96-well plate and add 100 μL / well of the diluted sample in ② and culture in a cell culture incubator at 37°C for 24 h;

[0112] ④ Aspirate the culture medium in the Vero cell 96-well plate and dry it at room temperature for 5 minutes; add immunostaining fixative along the wall at 100 μL / well and incubate at room temperature for 15 minutes. After incubation, aspirate the fixative and wash the cells in the plate twice with 200 μL / well of washing solution;

[0113] ⑤ Add dilution buffer to the cells in a 96-well plate at 200 μL / well and incubate at room temperature for 1 hour, then wash three times with washing solution;

[0114] ⑥ Add primary antibody working solution to the cell 96-well plate at 25 μL / well, incubate at 37°C for 1h±5min, and then wash 3 times with washing solution;

[0115] ⑦ Add the secondary antibody working solution to the cell 96-well plate at 25 μL / well, incubate at 37°C for 1h±5min, and then wash 3 times with washing solution; use ELISPOT instrument for detection; the detection parameters of ELISpot cytometer are shown in Table 1.

[0116] The dilution buffer used was a 1×PBS solution containing 1 wt.% BSA, 0.05% Proclin-300 by volume and 0.05% Tween-20 by volume, pH=7.4; the washing solution was a 1×PBS buffer solution, pH=7.4.

[0117] The LOD series samples are LOD samples diluted with dilution buffer to the concentrations listed in the analysis result table.

[0118] 5. Analysis results

[0119] The detection limit of the analyte in human urine is 5.00E+03pfu / mL

[0120] The detection limit of the analyte in the swab preservation fluid was 2.50E+03pfu / mL.

[0121] Table 7 Detection limit (urine) analysis results

[0122]

[0123] Table 8 Analytical results of detection limit (swab preservation solution)

[0124]

[0125]

[0126] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A method for detecting the infectivity of oncolytic viruses in urine and swab preservation fluid based on ELISpot technology, characterized in that: Vero cells were first cultured overnight in a transparent cell culture plate, and then the samples were transferred to a cell plate for culture. Immunofluorescence technology was then used to establish an indirect immunofluorescence method using rabbit anti-Vaccinia Virus PolyclonalAntibody as the primary antibody and goat anti-rabbit antibody labeled with Alexa FluorR 488 as the secondary antibody. The viral infectivity was determined by detecting the amount of fluorescent cells infected with the virus using an ELISpot instrument.

2. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 1, characterized in that: The following steps are involved: S1. Inoculate Vero cells into a 96-well cell plate and culture in an incubator overnight; S2, diluting the sample using cell growth medium; S3. When the confluence of Vero cells is ≥80%, the culture medium in the 96-well plate is discarded, and the sample diluted in step S2 is added and cultured in a cell culture incubator; S4, discard the culture medium in the Vero cell 96-well plate, dry at room temperature for 5 minutes; add immunostaining fixative along the wall and incubate, then remove the fixative and wash the cells in the wells with washing solution; S5, cells were added to the 96-well plate and incubated at room temperature in the dilution buffer, and then washed with washing solution; S6, add primary antibody working solution to the cell 96-well plate, and wash with washing solution after incubation; S7. Add the secondary antibody working solution to the 96-well cell plate, wash with washing solution after incubation; then use the ELISPOT instrument for detection.

3. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S1, 100 μL of Vero cell suspension was added to the 96-well plate. The concentration of the Vero cell suspension was 2.00×10 5 cells / mL, Vero cell number 2.00×10 4 The culture conditions were 5% CO2 and the culture temperature was 37°C.

4. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S2, the sample is urine or swab preservation fluid; the cell growth medium is a complete cell culture medium, 50 mL of fetal bovine serum and 5 mL of streptomycin-penicillin solution are added to 450 mL of 1640 culture medium; the sample is diluted to twice the volume using the cell growth medium.

5. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S3, the amount of diluted sample added is 100 μL / well, the culture temperature is 37° C., and the culture time is 24 h.

6. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S4, the fixative used is an immunostaining fixative, and the amount added is 100 μL / well; the incubation temperature is room temperature, the incubation time is 15 min, and the number of washes is 2 times.

7. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S5, the dilution buffer used is a 1×PBS solution containing 1 wt.% BSA, 0.05% Proclin-300 and 0.05% Tween-20, pH=7.4; the amount of dilution buffer added is 200 μL / well, the incubation time is ≥1h, and the number of washes is 3 times.

8. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S6, the primary antibody working solution used is prepared by using Vaccinia Virus Polyclonal Antibody and dilution buffer at a volume ratio of 1:400; the amount of primary antibody working solution added is 25 μL / well, the incubation temperature is 37° C., the incubation time is 1 h±5 min, and the number of washes is 3 times.

9. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: In step S7, the secondary antibody working solution used is Goat Anti-Rabbit lgG H&L, and is labeled with the fluorescent dye Alexa FluorR 488, and is prepared using a dilution buffer at a volume ratio of 1:200; the amount of the secondary antibody working solution added is 25 μL / well, the incubation temperature is 37° C., the incubation time is 1 h±5 min, and the number of washes is 3 times; the detection parameters of the ELISpot cytometer are: User Profiles: 2color fluorescence, Intensity: MIN:15, Size: MIN:

70.

10. The method for detecting oncolytic virus infectivity in urine and swab preservation fluid based on ELISpot technology according to claim 2, characterized in that: The detection limit for human urine is 5.00E+03pfu / mL, and the detection limit for swab preservation fluid is 2.50E+03pfu / mL.

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