Rapid determination method for virus content

Abstract

The invention discloses a rapid determination method of virus content, and belongs to the field of animal vaccine quality detection. The specific process comprises the following steps: (1) preparing virus antiserum; (2) diluting a to-be-detected virus solution, and culturing for 22-24 hours; (3) based on the prepared virus antiserum, determining the FAID50 of the virus to be detected, and then determining the TCID50 of the virus to be detected; (4) judging whether the content of the virus to be detected is qualified or not according to the absolute values A of the FAID50 and the TCID50 and the p values of the FAID50 and the TCID50; if the value A is less than or equal to 0.2 and the value p is more than or equal to 0.8, judging that the content of the virus to be detected is qualified, otherwise, judging that the virus is unqualified. The constructed method is low in detection cost and short in detection period, the whole detection period is 28 hours, the problem that the virus content is reduced due to the fact that the preservation time of a semi-finished product is prolonged is solved, and the content of a finished product is increased. In addition, when the virus content is detected, a virus standard substance does not need to be used, and the experiment cost is greatly reduced.

Description

Technical Field

[0001] The present invention belongs to the field of quality inspection of animal vaccines, and particularly relates to a rapid determination method for virus content. Background Art

[0002] The live vaccine against porcine pseudorabies virus is prepared by inoculating a susceptible cell with a attenuated strain of porcine pseudorabies virus for roller bottle culture, harvesting the cell culture, mixing it in a certain proportion, adding an appropriate amount of stabilizer, and then freeze-drying it under vacuum. It is used to prevent diseases caused by porcine pseudorabies virus infection.

[0003] The virus content measured by the cytopathic effect method is an important quality control index for the semi-finished and finished products of veterinary virus live vaccines, and is of great significance for the quality evaluation and clinical dosage of products. At present, veterinary virus biological products, including live virus vaccines, inactivated vaccines, etc., are all evaluated for the effectiveness of their production processes by the virus content measured by the cytopathic effect method. The virus content is also used as one of the standards for the activity and effectiveness during the preparation of the seed virus and semi-finished products. Therefore, the virus content is widely used in the manufacturing, inspection, and safety verification processes of veterinary biological products. It is very necessary to establish a rapid, accurate, and stable virus titration method. At present, the detection method for the virus content of veterinary virus live vaccines generally uses the cytopathic effect method to determine the virus content (TCID 50 )

[0004] Determination of virus content by the cytopathic effect method (TCID 50 ): Using suitable cells, diluting the virus solution to different gradients, inoculating the cells, and calculating the TCID 50 according to the number of cytopathic holes at different dilution degrees by the Reed-Muench method. This method has the following disadvantages: ① The detection period is long and requires 5 days; ② Since the maintenance time after cell inoculation with the virus is long, the natural apoptosis of cells affects the observation of the results of the cytopathic effect method.

[0005] Since after the virus solution of the virus live vaccine is harvested, it is necessary to determine the freeze-drying formula and ratio according to the virus content of the virus solution after the virus content result is determined. The virus is sensitive to the storage temperature and the virus is inactivated. Between 2~-20°C, the virus content in the virus solution drops rapidly. Shortening the time interval from virus solution harvest to freeze-drying is crucial for the production of virus live vaccines. Therefore, it is necessary to establish a new virus content detection method to shorten the time interval from virus solution harvest to freeze-drying, improve the virus content of the live vaccine, and reduce the production cost. Summary of the Invention

[0006] Aiming at the above deficiencies in the prior art, the present invention provides a rapid determination method for virus content.

[0007] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: A rapid method for determining virus content, comprising the following steps: (1) Prepare virus antiserum; (2) Dilute the virus liquid to be tested and culture it for 22 - 24 h; (3) Based on the prepared virus antiserum, determine the FAID of the virus to be tested by indirect immunofluorescence method 50 , and then use the cytopathic effect method to determine the TCID of the virus to be tested 50 ; (4) According to the absolute value A of FAID 50 and TCID 50 , and the p value of FAID 50 and TCID 50 , determine whether the content of the virus to be tested is qualified; If the A value ≤ 0.2 and the p value ≥ 0.8, it is determined that the content of the virus to be tested is qualified, otherwise it is unqualified.

[0008] Further, the virus antiserum in step (1) is animal antiserum.

[0009] Further, the virus antiserum in step (1) is porcine antiserum or rabbit antiserum.

[0010] Further, the preparation process of the virus antiserum in step (1) is: Take 10 7.5 ~10 8.5 TCID / mL attenuated virus of porcine pseudorabies virus and inoculate healthy susceptible pigs. The immunization program adopts three inoculations; 7 days after the third inoculation, collect a large amount of blood, separate the serum, inactivate and subpackage it to obtain the antiserum.

[0011] Further, the culture process in step (2) is: Take the virus liquid to be tested, perform 10-fold serial dilution, and inoculate it into a 96-well cell plate with monolayer ST cells. Inoculate 8 wells for each dilution, 100 μL / well. At the same time, set 4 wells of normal cell control, and continuously culture at 37 °C and 5% CO2 for 22 - 24 hours.

[0012] Further, the process of determining the FAID of the virus to be tested by indirect immunofluorescence method in step (3) is: 50 The process is: S1. Discard the liquid in the culture plate, add acetone + methanol pre-cooled at -15 °C, and fix it at -15 °C for 30 min; S2. After fixation, discard the fixing solution, and dry it in a fume hood or naturally for 30 min; S3. Add virus antiserum diluted with PBS containing 5% skim milk, 100 μL / well, and incubate at 37 °C for 90 min; S4. After the reaction is completed, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; S5. Add FITC-labeled goat anti-pig IgG diluted with PBS containing 5% skim milk, 100 μL per well, and incubate at 37 °C for 60 min; S6. After the reaction is completed, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; S7. Observe the fluorescence in each well under a fluorescence inverted microscope; record the luminescence of each well, and calculate the FAID by the Reed-Muench method according to the recorded results 50 。

[0013] Use of the above rapid method for determining virus content in constructing a virus content evaluation system.

[0014] Use of the above rapid method for determining virus content in the quality assessment of virus protectants.

[0015] Advantages of the present invention: The method constructed by the present invention has low detection cost and short detection period. The whole detection period is 28 hours, which avoids the problem of reduced virus content caused by the extended preservation time of semi-finished products and improves the content of finished products. In addition, when detecting the virus content, the present invention does not need to use virus standards, which greatly reduces the experimental cost. Detailed implementation manners

[0016] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the concept of the present invention are within the scope of protection.

[0017] Example 1 Preparation of antiserum Take attenuated virus of porcine pseudorabies virus (10 7.5 ~10 8.5 TCID 50 / mL) to inoculate healthy and susceptible pigs, and the immunization program is shown in Table 1.

[0018] Table 1 Immunization program for antiserum preparation

[0019] Seven days after the third immunization, collect a large amount of blood, separate the serum, inactivate and dispense it to prepare antiserum and measure the serum neutralizing antibody. The results are shown in Table 2.

[0020] Table 2 Results of antiserum neutralizing antibody determination

[0021] The obtained antiserum was stored at -75°C or below for future use.

[0022] Example 2 Detection of Virus Content in the Semi-finished Product of Swine Live Vaccine - Virus Liquid 1. Determination and Cultivation Take the semi-finished product of swine live vaccine - virus liquid stored at 2 - 8°C for different times, dilute it serially by 10-fold, and take 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Five dilutions were inoculated into a 96-well cell plate with ST cells grown to a monolayer, 8 wells were inoculated for each dilution, 100 μl / well, and at the same time, 4 wells of normal cell control were set. Each sample was replicated twice. One replicate was continuously cultured at 37°C and 5% CO2 for 22 - 24 h for indirect fluorescence staining, and the other replicate was continuously cultured at 37°C and 5% CO2 for 120 h to observe cytopathic effect.

[0023] 2. Indirect Fluorescence Staining After 22 - 24 h of culture, the prepared antiserum and FITC-labeled goat anti-pig IgG were used for staining, and the virus content FAID 50 was measured. The specific operation steps are as follows: ① Discard the liquid in the culture plate, add pre-cooled acetone + methanol (1:1) at -15°C, and fix at -15°C for 30 min; ② After fixation, discard the fixative, and dry it in a fume hood or naturally for 30 min; ③ Add the anti-pseudorabies virus serum diluted with PBS containing 5% skim milk powder, 100 μL / well, and incubate at 37°C for 90 min or at 2 - 8°C overnight; ④ After the reaction, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; ⑤ Add FITC-labeled goat anti-pig IgG diluted with PBS containing 5% skim milk powder, 100 μL / well, and incubate at 37°C for 60 min or at 2 - 8°C overnight; ⑥ After the reaction, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; ⑦ Observe the fluorescence of each well under a fluorescence inverted microscope; Result determination: The normal cell control wells should not show specific fluorescence, and the experiment is valid; Record the number of fluorescent wells at each dilution, and calculate FAID 50 according to the recorded results by the Reed-Muench method.

[0024] The principle of indirect immunofluorescent antibody staining method (FITC labeling) is based on the law of antigen-antibody reaction. The antiserum binds to the viral antigen in the cells, and the FITC-labeled goat anti-pig IgG binds to the antiserum, using it as a probe to detect porcine IgG in the cells. Under a fluorescence microscope, according to the fluorescence emitted by the formed complex, the source, nature, and location of the detected substance are determined. This method is simple, easy to perform, highly specific, fast, and convenient, and is commonly used for virus-specific staining. The present invention uses the indirect immunofluorescent antibody staining method to determine the content of the virus solution FAID 50 , and the results should be equivalent to those of the virus content TCID measured by the cytopathic effect method for samples stored at 2-8°C for different times 50 .

[0025] 3. Continuously culture for 120h by the cytopathic effect method and observe the cytopathic effect

[0026] (1)The calculations of both TCID 50 and FAID 50 adopt the Reed-Muench method. Based on the TICD 50 measured by the cytopathic effect method for virus solutions stored at different times and the FAID 50 measured by the indirect immunofluorescence method, the relationship between TCID 50 and FAID 50 is established as follows Compare the detection results FAID 50 of the content of the semi-finished product of the live swine vaccine - virus solution stored at 2-8°C for different times with the detection results TCID 50 of the virus content of the semi-finished product of the live swine vaccine - virus solution stored at 2-8°C for different times, and perform a T-test, with a P value ≥ 0.8 TCID 50 -FAID 50 |≤0.2

[0027] (2)Using the established relationship above, the virus content of the to-be-tested semi-finished product of the live swine vaccine - virus solution can be evaluated, and the judgment criteria are as follows For the FAID 50 of the virus solution stored at 2-8°C for different times and the TCID 50 of the virus solution stored at 2-8°C for different times, the T-test p ≥ 0.8, and TCID 50 -FAID 50 |≤0.2 is judged as qualified, otherwise it is judged as unqualified

[0028] 4. Application examples In the laboratory, three batches of semi-finished live porcine pseudorabies vaccines with different virus contents (high, medium, and low, see Table 5 for details, batch numbers: 20250401, 20250402, 20250403) were taken, and the specific preparation method is as follows: After the cells for vaccine production were resuscitated and appropriately proliferated by roller bottle culture, the virus liquid was prepared according to the following method. Preparation of porcine pseudorabies virus liquid: After the ST cells were amplified to the roller bottle scale, 7.5 mL of porcine pseudorabies virus liquid was inoculated by monolayer inoculation, and cultured at 37 °C and 5% CO2. The virus liquid could be harvested when 80% of the cells showed cytopathic effect. The harvested virus liquid was the porcine pseudorabies virus liquid. The porcine pseudorabies virus liquid was dispensed into 2 mL / vial and stored at 2 - 8 °C as a batch of semi-finished porcine pseudorabies virus.

[0029] After the harvested virus liquid was fully mixed, its virus content was measured (detected according to the appendix of the third part of the current edition of the Chinese Veterinary Pharmacopoeia). The prepared virus liquid was diluted with MEM cell culture medium respectively, and the virus content after dilution is shown in Table 5.

[0030] Table 5 Virus content measured by cytopathic effect method for each batch of porcine pseudorabies virus liquid stored at 2 - 8 °C for different times (TCID 50 ) and virus content measured by indirect immunofluorescence method FAID 50

[0031] TCID 50 and FAID 50 were both calculated by the Reed - Muench method, and the calculation formula is as follows: LgTCID 50 =- logarithm of the dilution of the sample equal to or higher than 50% + distance ratio × logarithm of the dilution factor Example: Table 6 Virus infection rate

[0032] Among them, the infection equal to or higher than 50% in Table 6 is 100%, and the infection lower than 50% in Table 6 is 0%. The logarithm of the virus dilution (10 -7 ) is -7. The logarithm of the dilution factor (1 / 10) is -1. The distance ratio was calculated according to the following formula: Distance ratio = (infection equal to or higher than 50% - 50%) / (infection equal to or higher than 50% - infection lower than 50%) = (100% - 50%) / (100% - 0%) = 0.50.

[0033] lgTCID 50 or lgFAID 50 =-7 + 0.50×(-1)= -7.5, then: TCID 50or FAID 50 =10 -7.50 / 0.1mL, indicating that the virus solution is 10 -7.50 After dilution, 0.1 mL of virus can be inoculated into each well to make 50% of the wells produce CPE or fluorescence. The virus content can be expressed as 10 7.50 TCID 50 or 10 7.50 FAID 50 or 10 8.50 TCID 50 / mL or 10 8.50 FAID 50 / mL. The TCID of the semi-finished virus solution stored at 2-8°C for different time periods was measured by cytopathic assay. 50 , and the FAID of the semi-finished virus solution stored at 2~8℃ for different time periods was measured by indirect immunofluorescence method 50 T-test was performed and the results are shown in Table 7.

[0034] Table 7 TCID of different batches of semi-finished live vaccines for pigs stored at 2-8℃ for different times 50 T-test (two-tailed, two-sample equal variance assumption) p-value between the value and the FAID50 value

[0035] The T-test calculation method is as follows: The virus content TCID was measured by cytopathic assay using the T.test formula using virus fluid stored at 2-8°C for different periods of time. 50 The data in the first column are the virus content FAID measured by indirect immunofluorescence method in virus liquid stored at 2~8℃ for different time periods. 50 For the second column of data, the p value is calculated using the two-tailed, two-sample equal variance assumption. The test results of the samples to be tested using the newly established method (FAID 50 ) and cytopathicity assay results TCID 50 The p-values were compared and the p-values were between 0.84 and 0.97. Based on this, the p-value ≥ 0.8 was determined to be qualified, otherwise it was unqualified.

[0036] The virus content of the semi-finished virus solution stored at 2-8°C for different time periods was measured by cytopathic assay (TCID) 50 Subtract the FAID of the virus content measured by indirect immunofluorescence method in the virus solution stored at 2-8℃ for different time periods 50 The absolute value of the obtained number is recorded as A.

[0037] Table 8 TCID of different batches of virus solution stored at 2-8℃ for different time periods 50 Value and FAID50 Absolute value A between values

[0038] The calculation method is as follows: The virus content results TCID measured by the cytopathic effect method for the virus solution stored at 2 - 8°C for different times 50 , respectively subtract the FAID measured by the indirect immunofluorescence method for the virus solution stored at 2 - 8°C for different times 50 . The absolute value of the obtained number is 10 0.0 ~10 0.2 / mL. Based on this, it is determined that the A value ≤ 10 0.2 / mL is qualified.

[0039] The method of the present invention has low detection cost, short detection cycle (the whole detection cycle is 28 hours), the detection result is not affected by the natural apoptosis of cells during long - term culture, avoiding interference. In addition, there is no need to use virus standard products, improving the experimental safety and further reducing the experimental cost. Finally, it should be noted that the above - mentioned specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A rapid determination method for virus content, characterized in that, It includes the following steps: (1) Prepare virus antiserum; (2) Dilute the virus solution to be tested and culture it for 22 - 24 h; (3) Based on the prepared virus antiserum, determine the FAID of the virus to be tested by indirect immunofluorescence assay 50 , and then determine the TCID of the virus to be tested by the cytopathic effect method 50 ; (4) According to FAID 50 and TCID 50 absolute value A, and FAID 50 and TCID 50 p-value to determine whether the virus content to be tested is qualified; If the A value ≤ 0.2 and the p value ≥ 0.8, it is determined that the content of the virus to be tested is qualified, otherwise it is unqualified.

2. The rapid determination method of virus content according to claim 1, characterized in that The virus antiserum in step (1) is animal antiserum.

3. The rapid determination method of virus content according to claim 2, characterized in that, The virus antiserum in step (1) is porcine antiserum or rabbit antiserum.

4. The rapid determination method of virus content according to claim 3, characterized in that The preparation process of the virus antiserum in step (1) is: Take 10 7.5 ~10 8.5 TCID / mL of attenuated pseudorabies virus of swine to inoculate healthy and susceptible pigs. The immunization program adopts three inoculations; seven days after the third inoculation, a large amount of blood is collected, the serum is separated, inactivated and sub-packaged to obtain antiserum.

5. The rapid determination method of virus content according to claim 1, wherein The culture process in step (2) is: Take the virus solution to be tested, perform 10-fold serial dilution, and inoculate the diluted solution into a 96-well cell plate with monolayer ST cells grown to confluence. Inoculate 8 wells for each dilution, 100 μL / well, and set 4 wells of normal cell control at the same time. Continuously culture at 37 °C and 5% CO2 for 22 - 24 hours.

6. The rapid determination method of virus content according to claim 1, characterized in that, In step (3), the FAID of the virus to be measured is determined by indirect immunofluorescence assay 50 The process is as follows: S1. Discard the liquid in the culture plate, add acetone + methanol pre-cooled at -15 °C, and fix at -15 °C for 30 min; S2. After fixation, discard the fixing solution, and dry it in a fume hood or naturally for 30 min; S3. Add virus antiserum diluted with PBS containing 5% skim milk, 100 μL / well, and incubate at 37 °C for 90 min; S4. After the reaction, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; S5. Add FITC-labeled goat anti-pig IgG diluted with PBS containing 5% skim milk, 100 μL / well, and incubate at 37 °C for 60 min; S6. After the reaction, wash the plate 5 times with PBS, and finally pat dry the residual liquid in the wells; S7. Observe the fluorescence in each well under a fluorescence inverted microscope; record the luminescence of each well, and calculate the FAID by the Reed-Muench method according to the recorded results 50 .

7. Use of the rapid determination method for virus content according to any one of claims 1 - 6 in constructing a virus content evaluation system.

8. Use of the rapid determination method for virus content according to any one of claims 1 - 6 in the quality assessment of virus protectants.

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