A method for detecting viral titer in TCID 50 and applications
By diluting the virus in a culture medium and inoculating cells, combined with microscopic observation and formula calculation, the TCID50 detection method was optimized, solving the problems of low detection accuracy and repeatability in existing technologies, and achieving high sensitivity and high repeatability of virus titer detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- MICRO SPECTRUM BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-05-22
- Publication Date
- 2026-07-21
Smart Images
Figure CN122428016A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analysis or testing, specifically to a TCID virus titer detection method. 50 Methods and applications. Background Technology
[0002] TCID 50 (50% tissue culture infection dose) is a viral titer assay that quantitatively assesses viral infectivity by precisely calculating the dilution required to induce cytopathic effects in 50% of cells after viral infection. However, TCID... 50 The detection results of this method are highly susceptible to interference from various factors, including cell seeding density, virus incubation time, and culture medium replenishment, which can affect the accuracy, repeatability, and efficiency of the detection method. Currently, TCID... 50 Current viral titer determination methods are not accurate enough for detecting many viruses, have poor repeatability, and cannot be applied in practical production and daily life. Therefore, it is crucial to develop a test method that meets detection standards in terms of specificity, linearity, repeatability, intermediate precision, accuracy, and robustness.
[0003] Chinese invention patent CN106940371A discloses a method for culturing and quantitatively detecting viral viruses. Under the action of polyethylene glycol, the virus settles, the cell membrane swells, and the cell's sensitivity to viral particles is increased, thus enhancing the TCID assay results. 50 During assays, optimizing antibody types and quality improves the staining rate and quality of immunofluorescence, thereby enhancing the accuracy of live virus quantification. However, this requires the introduction of polyethylene glycol, complicating the detection process. Chinese invention patent CN103343170B discloses a bovine viral diarrhea virus RT-PCR detection kit, which designs a pair of primers specifically amplifying BVDV, optimizes the RT-PCR reaction solution and conditions, uses a premixed reaction solution, reduces operational steps, shortens reaction time, and reduces the risk of contamination. However, it has a high detection limit and poor sensitivity. Summary of the Invention
[0004] To develop a test method that meets detection standards in terms of specificity, linearity, repeatability, intermediate precision, accuracy, and robustness, the first aspect of this invention provides a TCID virus titer detection method. 50 The method includes the following steps: Prepare the test sample of the virus: Dilute the virus to be tested using culture medium; Cultured cells; The diluted virus to be tested was added to the cells, and the cells were cultured further. Observe the cell status daily, and on the 6th day, observe the cells under a microscope, record the number of cell pores showing disease, and calculate TCID using the formula.50 value; TCID 50 = ds+ d; X = Log10 (Dilution of 100% infection) d = Log10 dilution coefficient s = Number of infected wells / Total number of inoculated wells.
[0005] This invention involves co-culturing cells with the test virus and culture medium, performing a series of dilution experiments on different viruses, and counting the number of infected wells for each concentration gradient. Statistical calculations yield the virus concentration that causes half-maximal cell infection. The method meets the standards for specificity, linearity, repeatability, intermediate precision, accuracy, and robustness. Compared to existing techniques that involve adding virus for infection followed by discarding and reculturing, this method significantly improves accuracy and sensitivity.
[0006] Under a microscope, each lesion showed specific cellular changes. Normal cells were adherent and spindle-shaped, while lesions resulted in rounded cells, detachment, empty spots at the bottom of the wells, and cell fusion.
[0007] In one embodiment, the culture medium is host cell culture medium DMEM; the host cell culture medium DMEM is a negative control group.
[0008] In one implementation method, the dilution of the virus to be tested is 10. 0 -10 -10 .
[0009] In one implementation method, the dilution of the virus to be tested is 10. 0 10 -1 10 -2 10 -3 10 -4 10 -6 10 -7 10 -8 10 -9 10 -10 .
[0010] In one embodiment, the cell culture density is (1-5) × 10⁻⁶. 3 Cells / pores.
[0011] In one embodiment, the cell culture density is 3 × 10⁻⁶. 3 Cells / pores.
[0012] As one implementation method, the cell culture conditions are 36-38°C and 4-6 vol% CO2.
[0013] In one embodiment, the method for culturing cells includes the following steps: seeding cells into a 96-well cell culture plate one day before the test, at a density of 3.0 × 10⁶ cells per well. 3 Cells were cultured at 37±1℃ in a 5±1% CO2 incubator.
[0014] In one embodiment, the cells are newborn bovine nasal turbinate cells (BT cells) or hamster kidney cells (BHK-21 cells).
[0015] As one implementation method, the method for adding the diluted test virus to cells includes the following steps: removing the culture medium from the 96-well cell culture plate, adding the test virus dilution to the cells in the 96-well plate from low to high concentration, adding 100 μL of dilution to each well, with 8 replicates (one column) for each dilution, the last column being a negative control, and adding host cell culture medium DMEM; as follows: Figure 1 As shown.
[0016] As one implementation method, the diluted virus to be tested is added to the cells for incubation and then supplemented with culture medium.
[0017] In one implementation method, the diluted test virus is added to the cells and incubated for 0-2 hours.
[0018] In one implementation, the diluted test virus is added to the cells and incubated for 1 hour.
[0019] In one embodiment, the culture medium is a complete DMEM medium, comprising 10 wt% FBS, 2 wt% antibiotics and 88 wt% DMEM basal medium.
[0020] In one implementation, the cells are cells that have proliferated for 20 generations or less.
[0021] As one implementation method, the virus titer detection TCID 50 The standard deviation of the method is <0.5.
[0022] A second aspect of the present invention provides a TCID virus titer detection method. 50 The method was applied to the detection of bovine viral diarrhea virus, vesicular stomatitis virus, and bovine parainfluenza virus type 3.
[0023] Compared with the prior art, the present invention has the following beneficial effects: (1) The virus titer detection TCID described in this invention 50 The method showed that adding the virus resulted in cytopathic effects in the wells, while the negative control group showed no cytopathic effects. This method is specific to a particular virus, and linear regression analysis showed a high linear correlation.
[0024] (2) The virus titer detection TCID described in this invention 50 The method involves inoculating cells with virus diluted in culture medium, resulting in a low minimum transfection concentration and a low limit of quantification for TCID detection. 50 The method has high sensitivity.
[0025] (3) The virus titer detection TCID described in this invention 50 The method exhibits good repeatability and intermediate precision, with a total standard deviation of <0.2, meeting the testing standards.
[0026] (4) The virus titer detection TCID described in this invention 50 The method uses cells from different passages for detection, and it has repeatability robustness against the same virus, with a total standard deviation of <0.2, which meets the detection standard.
[0027] (5) The virus titer detection TCID described in this invention 50 This method can be applied to the detection of viral titers for three types of viruses: bovine viral diarrhea virus, vesicular stomatitis virus, and bovine parainfluenza virus type 3. It can promote the in-depth development of virology research, accelerate the vaccine development process, and conduct more accurate evaluation of antiviral drugs. Attached Figure Description
[0028] Figure 1 TCID for virus titer detection in this invention 50 A schematic diagram of sample loading onto a cell culture plate.
[0029] Figure 2 The graph shows the linear regression equation for Example 1.
[0030] Figure 3 The graph shows the linear regression equation for Example 2.
[0031] Figure 4 The graph shows the linear regression equation for Example 3. Detailed Implementation
[0032] Example 1 A viral titer detection TCID 50 The method includes the following steps: Prepare the test sample of the virus: Dilute the virus to be tested using culture medium; Cultured cells; The diluted virus to be tested was added to the cells, and the cells were cultured further. Observe the cell status daily, and on the 7th day, observe the cells under a microscope, record the number of cell pores showing disease, and calculate TCID using the formula. 50 value; TCID50 = ds+ d; X = Log10 (Dilution of 100% infection) d = Log10 dilution coefficient s = Number of infected wells / Total number of inoculated wells.
[0033] The culture medium is host cell culture medium DMEM; the host cell culture medium DMEM is a negative control group.
[0034] The dilution of the virus to be tested was 10. 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9 10 -10 .
[0035] The cell culture density is 3 × 10⁻⁶. 3 Cells / pores.
[0036] The cell culture conditions were 37°C and 5 vol% CO2.
[0037] The cell culture method includes the following steps: one day before the test, the cells are seeded into 96-well cell culture plates, with 3.0 × 10⁶ cells per well. 3 Cells were cultured at 37±1℃ in a 5±1% CO2 incubator.
[0038] The cells in question are newly formed bovine nasal turbinate cells.
[0039] The virus to be tested was bovine parainfluenza virus type 3.
[0040] The method for adding the diluted test virus to cells includes the following steps: removing the culture medium from the 96-well cell culture plate; adding the test virus dilution buffer from low to high concentration to the cells in the 96-well plate, adding 100 μL of dilution buffer to each well, with 8 replicates per well (one column) for each dilution, the last column being a negative control; adding host cell culture medium DMEM; as follows: Figure 1 As shown.
[0041] The diluted virus to be tested was added to the cells and incubated for 1 hour, followed by replenishment of culture medium.
[0042] The culture medium is DMEM complete medium, which includes 10wt% FBS, 2wt% antibiotics and 88wt% DMEM basal medium.
[0043] The bispecific antibody is a penicillin-streptomycin solution.
[0044] Example 2 A viral titer detection TCID 50 The method and specific implementation are the same as in Example 1, except that the virus to be tested is bovine viral diarrhea virus. On day 6, cells were observed under a microscope, the number of cell pores showing lesions was recorded, and TCID was calculated using a formula. 50 value.
[0045] Example 3 A viral titer detection TCID 50 The method and specific implementation are the same as in Example 1, except that the virus to be tested is vesicular stomatitis virus. The cells are hamster kidney cells. On day 7, the cells are observed under a microscope, the number of cell pores showing lesions is recorded, and TCID is calculated using a formula. 50 value.
[0046] Performance testing 1. Specificity: The virus to be tested, diluted virus to be tested, and blank culture medium (negative control) were used for detection. Cytopathic effects were observed during the observation period. The results are shown in Table 1.
[0047] 2. Linearity and Limit of Quantitation: Experiments were conducted using different concentrations of the test virus, with each sample performed in triplicate, to determine linearity. Linear regression analysis yielded the linear equation. The results are shown in Table 1. The linear regression curves for Examples 1-3 are shown in [Table 1]. Figure 2-4 .
[0048] Table 1
[0049] 3. Precision: Repeatability: On the same day, experimenter A conducted two rounds of experiments using the virus to be tested as a sample, with three replicates in each round, to obtain the TCID. 50 Data were collected, and the standard deviation was calculated. Test results are shown in Table 3.
[0050] Intermediate precision: Using the virus to be tested as the test sample, and the experimenter, experiment date, pipette, etc. as variables, see Table 2 for the variable table, to obtain TCID. 50 Data were collected, and the standard deviation was calculated. Test results are shown in Table 3.
[0051] 4. Durability: On the same day, experimenter A used the virus to be tested as a sample and conducted experiments with cells at passages 12, 13, and 14, with three replicates per sample. The test results are shown in Table 3.
[0052] Table 2
[0053] Table 3
Claims
1. A viral titer detection TCID 50 The method is characterized by, Includes the following steps: Prepare the test sample of the virus: Dilute the virus to be tested using culture medium; Cultured cells; The diluted virus to be tested was added to the cells, and the cells were cultured further. Observe the cell status daily. On days 6-7, observe the cells under a microscope and record the number of pores showing disease. Calculate TCID using the formula. 50 value; TCID 50 = ds+ d; X = Log10 (Dilution of 100% infection) d = Log10 dilution coefficient s = Number of infected wells / Total number of inoculated wells.
2. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The dilution of the virus to be tested was 10. 0 -10 -10 .
3. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The cell culture density is (1-5)×10⁻⁶. 3 Cells / pores.
4. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The cell culture conditions are 36-38℃ and 4-6 vol% CO2.
5. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The diluted virus to be tested was added to the cells for incubation, and then culture medium was added.
6. The TCID for virus titer detection according to claim 5 50 The method is characterized by, The diluted virus to be tested was added to the cells and incubated for 0-2 hours.
7. The TCID for virus titer detection according to claim 5 50 The method is characterized by, The culture medium is DMEM complete medium, which includes 10wt% FBS, 2wt% antibiotics and 88wt% DMEM basal medium.
8. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The cells are at least one of newly formed bovine nasal turbinate cells or hamster kidney cells, and the cells have proliferated for no more than 20 generations.
9. The TCID for virus titer detection according to claim 1 50 The method is characterized by, The virus titer detection TCID 50 The standard deviation of the method is <0.
5.
10. A TCID method for detecting viral titers according to any one of claims 1-9 50 The application of the method is characterized by, It is used for the detection of bovine viral diarrhea virus, vesicular stomatitis virus, and bovine parainfluenza virus type 3.