Method for determining viral titers based on fluorescence microscopy observation combined with end-point dilution method

Abstract

The application provides a method for determining virus titer based on fluorescence microscope qualitative observation and end-point dilution method, comprising the following steps: inoculating host cells into culture containers for cell adhesion; gradiently serially diluting a virus sample to be detected carrying a fluorescence reporter gene; adding the virus diluent into each well of the culture containers, and setting multiple parallel sample cultures for each dilution; observing whether positive cells expressing the fluorescence reporter gene exist in each well by using a fluorescence microscope; recording the number of fluorescence positive wells in each dilution, and calculating 50% tissue culture infective dose (TCID50) by using the end-point dilution method to obtain the infection titer of the virus. The method provided by the application has low equipment requirement, simple operation and low cost, and can realize accurate determination of virus titer by using only an ordinary fluorescence microscope, and is suitable for titer determination and neutralizing antibody detection of various viruses carrying fluorescence reporter genes such as HPV pseudovirus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for determining viral titers based on qualitative observation using fluorescence microscopy combined with an endpoint dilution method. Background Technology

[0002] Human papillomavirus (HPV) is a common epitheliotropic virus closely associated with the development of various malignant tumors, including cervical cancer. HPV pseudoviruses (PsV) mimic the HPV viral capsid structure on their surface and encapsulate reporter plasmids carrying reporter genes (such as green fluorescent protein GFP). They are widely used in HPV neutralizing antibody detection, vaccine efficacy evaluation, and research on viral infection mechanisms. Accurate determination of the infection titer of HPV pseudoviruses is fundamental to these studies and applications.

[0003] Currently, the commonly used methods for determining HPV pseudovirus titers mainly include the following: (a) Flow cytometry detection method Flow cytometry allows for high-speed, quantitative analysis of single cells. By detecting the expression of reporter genes (such as GFP) or specific fluorescent markers within infected cells, the proportion of positive cells can be directly calculated, thereby estimating the number of infectious viral particles. While this method achieves precise quantification at the single-cell level, flow cytometry equipment is expensive, complex to operate, and requires a high number and condition of cells, making it unsuitable for laboratories with limited resources.

[0004] (ii) qPCR method Plasmid DNA is extracted from infected cells, and the copy number is detected by quantitative PCR to indirectly reflect the infection titer. This method has high sensitivity, but it requires cumbersome steps such as cell lysis and DNA extraction, which is time-consuming. In addition, qPCR instruments are expensive and prone to introducing operational errors.

[0005] (III) Fluorescent microplate reader detection method Cells are lysed after infection, and the fluorescence intensity is detected. The infection units are estimated using a standard curve. This method is relatively simple to operate, but it still requires cell lysis, and the quantification of fluorescence signals depends on the accuracy of the standard curve.

[0006] In summary, the existing methods for determining HPV pseudovirus titers have the following shortcomings: (1) High equipment cost, with expensive instruments such as flow cytometers and qPCR instruments; (2) Complex operation, requiring steps such as cell lysis, DNA extraction, and fluorescent labeling, which is time-consuming and prone to introducing errors; (3) Sensitive to cell quantity, as flow cytometry requires a large number of cells and has high requirements for cell state; (4) Not suitable for high-throughput screening, as existing methods are inefficient when processing large numbers of samples.

[0007] Therefore, there is an urgent need in this field for a method for determining HPV pseudovirus titers that is easy to operate, requires low equipment, is inexpensive, and is suitable for high-throughput screening. Summary of the Invention

[0008] This invention aims to at least partially solve one of the technical problems in related technologies. Existing methods for determining viral titers suffer from drawbacks such as high equipment costs, complex operation, reliance on subjective judgment by CPEs, and the inability to count viral titers using fluorescence microscopes. This invention provides a method for determining viral titers based on qualitative observation using fluorescence microscopy combined with the endpoint dilution method. By combining qualitative observation (determining positive / negative results) using fluorescence microscopy with statistical quantification using the endpoint dilution method (calculating TCID50), this invention cleverly circumvents the technical bottleneck of counting viral titers using fluorescence microscopy. It enables accurate determination of viral titers using only a common inverted fluorescence microscope, and has advantages such as simple operation, low cost, objective results, and suitability for high-throughput screening.

[0009] Specifically, the present invention provides the following technical solution: This invention provides a method for determining viral titers based on qualitative observation using fluorescence microscopy combined with an endpoint dilution method, comprising: (1) Inoculate the host cells into a culture container and culture them to allow the cells to adhere to the container; (2) The virus sample carrying the fluorescent reporter gene was serially diluted according to the gradient to obtain several virus dilutions with different dilutions; (3) Add the virus dilution obtained in step (2) to each well of the culture container described in step (1), and set up multiple parallel samples for each dilution for culture; (4) Set up positive control wells and negative control wells, and use a fluorescence microscope to observe whether there are positive cells expressing fluorescent reporter genes in each well; (5) Record the number of fluorescent positive wells in each dilution, calculate the 50% tissue culture infection dose using the endpoint dilution method, and thus obtain the infection titer of the virus.

[0010] This invention provides a method for determining viral titers based on qualitative observation using fluorescence microscopy combined with an endpoint dilution method. Viral samples are serially diluted and then used to infect host cells. Since viral infection events between cells are random, at appropriate dilutions, some wells will be infected (showing positive fluorescence), while others will remain uninfected (negative fluorescence). The presence of fluorescently positive cells in each well is qualitatively determined using fluorescence microscopy (rather than precisely counting the number of positive cells). The number of positive wells at each dilution is recorded, and the TCID50 is calculated based on the statistical principles of the endpoint dilution method. This combination of qualitative observation and statistical quantification fundamentally overcomes the inherent limitation of fluorescence microscopy's inability to count cells. The method provided by this invention does not require complex equipment such as flow cytometers or qPCR instruments; titer determination can be completed using only a common inverted fluorescence microscope. It offers advantages such as ease of operation, low cost, objective results, and suitability for high-throughput screening.

[0011] According to an embodiment of the present invention, the host cell in step (1) is 293TT cells, HeLa cells, or HaCaT cells. 293TT cells are the most commonly used host cells for HPV pseudovirus titer assays, possessing advantages such as easy transfection, rapid growth, and high susceptibility to HPV pseudovirus infection. HeLa cells and HaCaT cells can also be used for HPV pseudovirus infection experiments, and different cell lines can be selected according to experimental requirements. According to a preferred embodiment, the host cell is 293TT cells.

[0012] According to an embodiment of the present invention, the seeding amount of cells in step (1) is 1.0 × 10⁻⁶. 4 ~4.0×10 4 Cells / well. Inoculum size less than 1.0 × 10⁻⁶. 4 When the inoculum size is 10 cells / well, the cell density is too low, and the cells are sparsely distributed in the 96-well plate, which is not conducive to intercellular signal transduction and maintaining normal growth. Furthermore, the fluorescence signal after viral infection is weak, which is not conducive to qualitative interpretation under a fluorescence microscope. Inoculation density exceeding 4.0 × 10⁶ cells / well results in a low cell density and sparse cell distribution in the 96-well plate, which is detrimental to intercellular signal transduction and maintaining normal growth. 4 At a cell / well density of 1.0 × 10⁶ cells, excessive cell density can lead to premature apoptosis due to insufficient nutrients during culture. Furthermore, overly dense cell populations can cause overlapping fluorescence signals, interfering with accurate interpretation of positive / negative results. Therefore, the inoculum size should be controlled at 1.0 × 10⁶ cells / well. 4 ~4.0×10 4 The spacing between cells / well ensures both a uniform monolayer of cells within the 96-well plate (facilitating fluorescence microscopy observation) and clear visibility of fluorescence signals after viral infection. According to a specific embodiment of the invention, the cell seeding density is 1.5 × 10⁻⁶ cells / well. 4 Under these conditions, cells adhere evenly to the well, exhibit optimal growth, and provide the clearest interpretation of fluorescence signals.

[0013] According to an embodiment of the present invention, the fluorescent reporter gene is selected from at least one of green fluorescent protein, red fluorescent protein, or yellow fluorescent protein.

[0014] According to an embodiment of the present invention, the fluorescence microscope described in step (4) is an inverted fluorescence microscope equipped with a green fluorescent protein detection channel. An inverted fluorescence microscope is the preferred device for observing adherent cultured cells. Its objective lens is located below the stage, allowing direct observation of adherent cells at the bottom of the culture vessel without the need for slide preparation, making operation extremely simple. Equipped with a GFP detection channel (typical excitation wavelength 450–490 nm, emission wavelength 500–550 nm), the green fluorescence of GFP-positive cells can be clearly observed. The aforementioned inverted fluorescence microscope includes, but is not limited to, at least one of the Ts2 fluorescence microscope, Olympus IX73, and Nikon Ti2.

[0015] According to an embodiment of the present invention, the serial dilution in step (2) is a 10-fold gradient dilution, with a dilution range of 10. - ¹ to 10 -9 Each dilution should have 4-8 replicates. 10-fold serial dilution is the most commonly used dilution method in endpoint dilution, as it is simple to operate and calculate. Dilution range (10... - ¹ to 10 -9 This covers the titer range of the vast majority of viral samples, ensuring that the proportion of positive wells exceeds the critical 50% range at appropriate dilutions, thus guaranteeing the accuracy of TCID50 calculations. Setting 4 to 8 replicates (e.g., 4 replicates) ensures the statistical reliability of the proportion of positive wells at each dilution.

[0016] According to an embodiment of the present invention, the culture time in step (3) is 48 to 96 hours. Culture time is a key parameter affecting the intensity of the fluorescence signal and the accuracy of positive well interpretation. When the culture time is less than 48 hours, the virus may not have completed a sufficient infection and replication cycle, resulting in insufficient expression of the fluorescent reporter gene, weak fluorescence signal, and a high likelihood of false negatives (i.e., wells that are actually infected are mistakenly judged as negative due to weak fluorescence). When the culture time is longer than 96 hours, cells may experience contact inhibition or even apoptosis due to overgrowth, and fluorescent proteins may accumulate excessively in infected cells, leading to fluorescence signal diffusion and interfering with the interpretation of adjacent wells. The culture time range of 48 to 96 hours ensures that the fluorescent reporter gene has sufficient time to express to a detectable level, while avoiding interference from cell deterioration on interpretation. 72 hours is particularly preferred, as the fluorescence signal is strongest and most stable at this time point, and the positive / negative contrast is most pronounced.

[0017] According to an embodiment of the present invention, the endpoint dilution method in step (5) is either the Reed-Muench method or the Karber method. The Reed-Muench method and the Karber method are two classic statistical methods for calculating TCID50 using endpoint dilution. The Reed-Muench method determines the 50% infection endpoint by calculating the cumulative positive and negative percentages, and is suitable for various dilution designs; the Karber method is based on the linear relationship between dilution and the positive percentage, making the calculation simpler. Both methods can obtain accurate TCID50 values, and the choice can be made according to experimental practices.

[0018] According to an embodiment of the present invention, the calculation formula of the Reed-Muench method is as follows: Distance ratio = (positive rate above 50% - 50%) / (positive rate above 50% - positive rate below 50%); log TCID50 = logarithm of dilution greater than 50% + distance ratio × logarithm of dilution factor; TCID50 (per mL) = 10^(logarithm of endpoint dilution) × (1 / inoculation volume mL).

[0019] According to an embodiment of the present invention, the virus is at least one of HPV pseudovirus, lentivirus, adenovirus, adeno-associated virus, or vesicular stomatitis virus.

[0020] The provided method can also be used to determine the antiviral and virus-removing performance of household appliances and materials, as well as for water quality testing and evaluation of virus killing effects.

[0021] The method provided by this invention ingeniously combines qualitative observation by fluorescence microscopy (determining only positive / negative) with statistical quantification by endpoint dilution, completely circumventing the inherent limitation of fluorescence microscopy in accurately counting positive cells. This invention has at least the following beneficial effects: (1) Low equipment requirements: Only a regular fluorescence microscope is needed to complete the detection, without the need for expensive equipment such as flow cytometers and qPCR instruments, which significantly reduces the detection cost and makes virus titer determination possible in conventional laboratories with limited resources; (2) Simple operation: No need to lyse cells, extract DNA, use fluorescent dyes or perform complicated sample pretreatment. Positive wells can be determined by directly observing the fluorescence signal in living cells through a fluorescence microscope, which reduces the number of operation steps and error introduction links. (3) Qualitative observation and quantitative calculation: Positive wells are identified qualitatively by fluorescence microscopy, and the virus titer is accurately quantified by combining the classic biostatistical method (TCID50), which eliminates the subjective judgment error in the traditional CPE observation method and makes the results of different operators comparable; (4) Applicable to viruses that do not produce CPE: This invention does not rely on the observation of cytopathic effects, but directly determines infection through the expression of fluorescent reporter genes. Therefore, it is particularly applicable to viruses such as HPV pseudoviruses that do not produce typical CPE.

[0022] (5) Low cost and high throughput: The 96-well plate can process multiple dilutions and multiple replicates at the same time, which is suitable for applications that require high-throughput screening, such as vaccine development and neutralizing antibody detection. (6) Real-time observation of live cells: No need to fix or lyse cells, observation can be performed multiple times during the culture process to achieve dynamic tracking of the virus infection process.

[0023] (7) Reliable methodological verification: After comparison with the industry standard fluorescent spot method, the logarithmic value of HPV pseudovirus titer measured by the method of this invention is not significantly different from that of the fluorescent spot method (P>0.05), indicating that this method can obtain accurate results comparable to professional analytical instruments while significantly reducing equipment costs. Attached Figure Description

[0024] Figure 1 The image shows a fluorescence micrograph of GFP-positive cells under a fluorescence microscope, provided according to an embodiment of the present invention. The cells are 293TT cells (GFP-positive) infected with HPV pseudovirus.

[0025] Figure 2 Fluorescence micrograph of negative control cells under a fluorescence microscope provided according to an embodiment of the present invention; these are 293TT cells (negative control) that are not infected with HPV pseudovirus.

[0026] Figure 3 This is a flowchart of the TCID50 calculation process of the present invention (Reed-Muench method).

[0027] Figure 4 The bar chart shows a comparison of the logarithmic values ​​of the HPV pseudovirus particle titer determined by the method of this invention and by fluorescent spot determination (ns indicates no significant difference). Detailed Implementation

[0028] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0029] This invention provides a method for determining viral titers based on qualitative observation using fluorescence microscopy combined with an endpoint dilution method, comprising: (1) Inoculate the host cells into a culture container and culture them to allow the cells to adhere to the container; (2) The virus sample carrying the fluorescent reporter gene was serially diluted according to the gradient to obtain several virus dilutions with different dilutions; (3) Add the virus dilution obtained in step (2) to each well of the culture container described in step (1), and set up multiple parallel samples for each dilution for culture; (4) Set up positive control wells and negative control wells, and use a fluorescence microscope to observe whether there are positive cells expressing fluorescent reporter genes in each well; (5) Record the number of fluorescent positive wells in each dilution, calculate the 50% tissue culture infection dose using the endpoint dilution method, and thus obtain the infection titer of the virus.

[0030] The method provided by this invention is not only applicable to HPV pseudoviruses, but also generally applicable to various viruses carrying fluorescent reporter genes, including but not limited to lentiviruses, adenoviruses, adeno-associated viruses (AAV), and vesicular stomatitis viruses (VSV). All of these viruses can infect adherent mammalian cells in vitro and express fluorescent reporter genes after infection, producing fluorescent signals detectable by a fluorescence microscope. These viruses can all carry fluorescent reporter genes; can all infect adherent mammalian cells and express exogenous genes; and after infection, positive and negative wells can be clearly distinguished by fluorescence signals, meeting the binary interpretation requirements of the endpoint dilution method. The statistical principle of the endpoint dilution method does not depend on the specific type of virus; as long as it can be clearly determined whether there are fluorescently positive cells in each well, the method of this invention can be used to calculate TCID50. Furthermore, the accuracy and reliability of the method of this invention have been verified through comparative experiments with the fluorescent dot method.

[0031] According to the preferred embodiment, the virus mentioned is a pseudovirus. Traditional endpoint dilution methods are completely ineffective against pseudoviruses. Pseudoviruses do not produce cytopathic effects (CPE), and traditional endpoint dilution methods rely on CPE to determine positive wells, thus making them unsuitable for pseudovirus titer determination. This invention uses fluorescence signals instead of CPE as the interpretation indicator, fundamentally solving this technical problem. Theoretically, one infectious unit (1 TCID50) of a pseudovirus corresponds to one fluorescently positive cell. The criteria for determining a positive well are clear—as long as a fluorescently positive cell is present in the well, it is a positive well, eliminating the need for precise counting and overcoming the technical bottleneck of fluorescence microscopy's inability to accurately count cells. Furthermore, pseudoviruses are widely used in neutralizing antibody detection, vaccine evaluation, and drug screening, involving parallel testing of a large number of samples. Existing methods rely on expensive equipment such as flow cytometers and enzyme-linked immunospot analyzers, which are difficult to meet high-throughput requirements. This invention only requires a regular fluorescence microscope and a 96-well plate to achieve high-throughput detection, significantly reducing costs. Pseudoviruses can be directly observed as live cells under a regular fluorescence microscope without the need for fixation, lysis, or inactivation, simplifying operation and reducing errors.

[0032] The provided method is suitable for determining the infectivity of HPV pseudoviruses, such as titer calibration of packaged HPV pseudoviruses to ensure quality control across different batches; and for detecting HPV neutralizing antibodies, for example, in vaccine efficacy evaluation, by detecting the neutralizing ability of serum samples against HPV pseudoviruses to assess vaccine protective effects; or for antiviral drug screening: in the development of anti-HPV drugs, by measuring viral titer changes before and after drug treatment to evaluate the antiviral activity of the drug. It can also be used to determine the antiviral and virus-removing performance of household appliances and materials, as well as for water quality testing and evaluation of virus-killing effects.

[0033] The method provided by this invention only needs to determine whether there are fluorescent positive cells in each well, without the need to accurately count the positive cells.

[0034] According to a specific embodiment, the mammalian cells are selected from at least one of 293TT cells, HeLa cells, or HaCaT cells.

[0035] According to a specific implementation method, the cell seeding concentration is 1.5 × 10⁻⁶. 4 Each cell.

[0036] The microscope used can be used to obtain the infection titer of HPV pseudoviruses.

[0037] The calculation formula for the Reed-Muench method is as follows: Distance ratio = (positive rate above 50% - 50%) / (positive rate above 50% - positive rate below 50%); log TCID50 = logarithm of dilution greater than 50% + distance ratio × logarithm of dilution factor; TCID50 (per mL) = 10^(logarithm of endpoint dilution) × (1 / inoculation volume mL).

[0038] The fluorescent reporter gene can be selected from any one of green fluorescent protein (GFP), red fluorescent protein (mCherry), or yellow fluorescent protein (YFP), with GFP being preferred.

[0039] The technical solution of the present invention will be described below through specific embodiments. It should be noted that these embodiments are only used to facilitate understanding by those skilled in the art and should not be regarded as a limitation on the scope of protection of the present invention. Unless otherwise specified, the instruments or reagents used in the embodiments can be obtained commercially.

[0040] Example 1 Example 1 describes the determination of HPV pseudovirus titers using the following method. The pseudovirus particle (PsV, Pseudovirus) mimics the HPV viral capsid structure on its surface and encapsulates a reporter plasmid carrying the green fluorescent protein (GFP) gene. After infecting host cells, the GFP gene within the pseudovirus is expressed, causing the infected cells to emit green fluorescence. Fluorescently positive wells (i.e., GFP-positive wells) are qualitatively detected using a Ts2 microscope, and the TCID50 is calculated using the Reed-Muench method or the Karber method, which is then converted into the infection titer.

[0041] The experimental materials used are as follows: Cells: 293TT cells (passages 3-15); Virus: HPV pseudovirus (carrying the GFP reporter gene); Culture medium: DMEM complete medium (containing 10% fetal bovine serum, final concentration of 250 μg / mL hygromycin); The instruments used were: a Ts2 inverted fluorescence microscope (excitation wavelength 488nm, emission wavelength 509nm) and a CO2 cell culture incubator.

[0042] The experimental steps include: 1) Cell resuscitation 293TT cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and seeded into T75 culture flasks containing 15 mL of complete cell culture medium (containing 250 μg / mL hygromycin). The flasks were then incubated at 37°C in a 5% CO2 incubator.

[0043] 2) Cell passage When the cells have adhered to the culture vessel and grown to approximately 80% confluence, they are passaged. The culture medium in the culture flask is discarded, and the cells are washed twice with 5 mL of PBS. 3 mL of trypsin is added for digestion for 1-2 minutes, and 5 mL of complete cell culture medium (containing 250 μg / mL hygromycin) is added to stop the digestion. The cells are repeatedly pipetted to prepare a single-cell suspension. The suspension is then passaged at a ratio of 1:3, and the volume of culture medium is increased to 15 mL. The cells are then placed in a 37°C, 5% CO2 incubator for further culture.

[0044] 3) Cell plating 293TT cells in the logarithmic growth phase (passages 3-15) were collected, counted, and the cell density was adjusted. The cells were then seeded into 96-well cell culture plates at 100 μL per well, with a seeding volume of approximately 1.5 × 10⁻⁶ cells / well. 4 One cell per well was placed in a 37°C, 5% CO2 incubator for 4 hours to allow the cells to adhere fully.

[0045] 4) Virus dilution The HPV pseudovirus stock solution was serially diluted 10-fold with complete culture medium to obtain 10 -1Up to 10 -9 There are a total of 9 dilutions of the virus.

[0046] 5) Infection Add 100 μL of virus dilution buffer at each dilution to each well of the 96-well plate described in step 3), with four replicates per dilution. Also include negative control wells (without virus). Incubate at 37°C in a 5% CO2 incubator for 72 hours.

[0047] 6) Fluorescence observation Observation was performed well by well using a Ts2 inverted fluorescence microscope (excitation wavelength 488 nm, emission wavelength 509 nm).

[0048] As can be seen, GFP-positive cells exhibit green fluorescence under a fluorescence microscope (e.g., Figure 1 As shown), the negative control well showed no green fluorescence (as shown). Figure 2 (As shown). Record the number of GFP-positive wells in each dilution, as shown in Table 1 below. Since 10 -1 and 10 -2 All wells were expected to be positive at each dilution. This data is only used to confirm the effectiveness of the dilution gradient. For simplicity, Table 1 only shows 10. -3 ~10 -9 The representative results show that the complete dilution gradient was actually used in the experiment.

[0049] Table 1

[0050] Wherein, C represents the negative control well, S10 -3 ~S10 -9 This indicates a virus dilution of 10. -3 ~10 -9 Among them, numbers 1-4 are equivalent to one experiment, numbers 5-8 are equivalent to one experiment, and numbers 9-12 are equivalent to one experiment.

[0051] 7) Data processing Data was compiled based on the recorded number of positive wells (as shown in Table 2 below), and the proportion of positive wells and the cumulative number of positive / negative wells for each dilution were calculated (the data in the table are exemplary and conform to the Reed-Muench cumulative rule). The cumulative values ​​were added sequentially from the highest concentration (lowest dilution) to the lowest concentration (highest dilution), with the cumulative number of positive wells added from bottom to top and the cumulative number of negative wells added from top to bottom; the lesion rate (i.e., the cumulative positive proportion) = cumulative number of positive wells / (cumulative number of positive wells + cumulative number of negative wells) × 100%.

[0052] Table 2 Data Compilation Table

[0053] 8) TCID50 calculation The 50% endpoint dilution was calculated using the Reed-Muench method: First, the dilutions corresponding to positive rates above 50% and below 50% were determined according to Table 2. Table 2 shows that the dilution for positive rates above 50% is 10. 6 (Disease rate 80%), dilutions below 50% are 10. 7 (Lesion rate 20%).

[0054] Then, calculate according to the following formula: Distance ratio = (positive rate above 50% - 50%) / (positive rate above 50% - positive rate below 50%); log TCID50 = logarithm of dilution greater than 50% + distance ratio × logarithm of dilution factor; TCID50 (per mL) = 10^(logarithm of endpoint dilution) × (1 / inoculation volume mL).

[0055] Let's take the data in Table 2 as an example for specific calculations: Distance ratio = (80% - 50%) / (80% - 20%) = 30% / 60% = 0.5; log TCID50 (per 0.1 mL) = 6 + 0.5 × 1 = 6.5, meaning the endpoint dilution is 10. 6.5 ; TCID50 / 0.1mL = 10 6.5 (approximately 3.16 × 10) 6 ); Converted to per mL: TCID50 / mL = 10 6.5 × (1 / 0.1) = 10 6.5 × 10 = 10 7.5 ≈ 3.16×10 7 .

[0056] This allows for the accurate determination of the 50% tissue culture infection dose of HPV pseudovirus in that batch, a value that directly reflects the viral infection titer. The calculation flowchart is shown below. Figure 3 As shown.

[0057] Example 2 To verify the accuracy and reliability of the method of this invention, a comparative experiment was conducted between this method (qualitative observation by fluorescence microscopy combined with the Reed-Muench method) and the fluorescent spot method used by the supplier (using an ImmunoSpot S6 Universal M2 fluorescence enzyme-linked immunospot analyzer to calculate the viral particle titer by detecting the number of fluorescent spots in the lesion wells). The instruments and specifications used in both methods are shown in Table 3. The titers of the same batch of HPV pseudovirus particles (catalog number DD1911) were determined, and the results are shown in Table 4.

[0058]

[0059] Table 4. Results of HPV pseudovirus particle titer determination by two methods

[0060] The logarithmic titers obtained by the two methods were analyzed for significance using Graphpad Prism 10.1.2 software (see [link to analysis]). Figure 4 The results showed no significant difference between the logarithmic titer obtained by the fluorescent spot method and the logarithmic titer obtained by the method of the present invention (P > 0.05). This indicates that the results obtained by the method of the present invention are highly consistent with the industry-recognized fluorescent spot method, verifying the accuracy and reliability of the method of the present invention.

[0061] The above verification results fully demonstrate that the method for determining viral titers based on qualitative observation using fluorescence microscopy combined with endpoint dilution established in this invention can obtain accurate results comparable to those of professional fluorescent spot analyzers, while significantly reducing equipment costs and operational complexity. It is suitable for routine titer determination of viruses carrying fluorescent reporter genes, such as HPV pseudoviruses.

[0062] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for determining viral titer based on qualitative observation using fluorescence microscopy combined with an endpoint dilution method, characterized in that, include: (1) Inoculate the host cells into a culture container and culture them to allow the cells to adhere to the container; (2) The virus sample carrying the fluorescent reporter gene was serially diluted according to the gradient to obtain several virus dilutions with different dilutions; (3) Add the virus dilution obtained in step (2) to each well of the culture container described in step (1), and set up multiple parallel samples for each dilution for culture; (4) Set up positive control wells and negative control wells, and use a fluorescence microscope to observe whether there are positive cells expressing fluorescent reporter genes in each well; (5) Record the number of fluorescent positive wells in each dilution, calculate the 50% tissue culture infection dose using the endpoint dilution method, and thus obtain the infection titer of the virus.

2. The method according to claim 1, characterized in that, The host cells mentioned in step (1) are 293TT cells, HeLa cells, or HaCaT cells.

3. The method according to claim 1, characterized in that, The seeding amount of cells in step (1) is 1.0 × 10⁻⁶. 4 ~4.0×10 4 Cells / well 4. The method according to claim 1, characterized in that, The fluorescent reporter gene is selected from at least one of green fluorescent protein, red fluorescent protein, or yellow fluorescent protein.

5. The method according to claim 1, characterized in that, The fluorescence microscope mentioned in step (4) is an inverted fluorescence microscope equipped with a green fluorescent protein detection channel.

6. The method according to claim 1, characterized in that, The serial dilutions described in step (2) are 10-fold serial dilutions, with a dilution range of 10. -1 Up to 10 -9 Each dilution has 4 to 8 replicates.

7. The method according to claim 1, characterized in that, The incubation time in step (3) is 48 to 96 hours.

8. The method according to claim 1, characterized in that, The endpoint dilution method mentioned in step (5) is the Reed-Muench method or the Karber method.

9. The method according to claim 8, characterized in that, The calculation formula for the Reed-Muench method is as follows: Distance ratio = (positive rate above 50% - 50%) / (positive rate above 50% - positive rate below 50%); log TCID50 = logarithm of dilution greater than 50% + distance ratio × logarithm of dilution factor; TCID50 (per mL) = 10^(logarithm of endpoint dilution) × (1 / inoculation volume mL).

10. The method according to claim 1, characterized in that, The virus is at least one of HPV pseudovirus, lentivirus, adenovirus, adeno-associated virus, or vesicular stomatitis virus.

Images