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Method for determining titer of reovirus type 3 through tissue half infection method staining

A reovirus, half-infection technology, applied in the field of biomedicine, can solve problems that have not been discovered yet, and achieve the effect of reducing errors, strong repeatability, and good stability

Pending Publication Date: 2022-03-01
SUZHOU YAOMING KANGDE INSPECTION TESTING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Through the search of Chinese and English patents, no clear patents have been found for the determination of Reo-3 titer using the tissue half-infection method

Method used

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  • Method for determining titer of reovirus type 3 through tissue half infection method staining
  • Method for determining titer of reovirus type 3 through tissue half infection method staining
  • Method for determining titer of reovirus type 3 through tissue half infection method staining

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Tissue half infection method staining and determination of Reo-3 virus titer specific implementation method

[0033] First, after the Reo-3 virus was thawed, after ultrasonication and filtration, a 10-fold serial dilution was made according to the actual situation. The virus dilution was EMEM medium with HEPES as a buffer, and the concentration of HEPES was 0.25mol / L;

[0034] Next, add MEM medium in the cell culture 96-well plate, the fetal calf serum content of the MEM medium is 8%, inoculate LLC-MK2 cells in the cell culture 96-well plate, inoculate about 4× in each cell well 104 cells at 37°C, 5% CO 2 Carry out cell culture under culture conditions, observe the cells on the second day, and proceed to subsequent operations when the cell confluence is 70-100%, and the number of inoculated LLC-MK2 cells can be adjusted within a reasonable range to make the cell confluence reach within two days 70-100% can be followed up;

[0035] Again, use the prepared Reo...

Embodiment 3

[0040] Example 3 The same operator carried out the comparison of Reo-3 virus infection LLC-MK2 cell experiment at different times

[0041] According to the operation process of Example 1, the experimental operator Operator1 carried out the titer determination experiments Run1 and Run3 of the Reo-3 virus on the first day and the second day respectively. Repeatedly, after serial dilution of the sample, do 8 multiple hole experiments for each dilution, exclude samples whose virus concentration is lower than the detection limit, and select four samples for result analysis, the results are as follows: image 3 shown.

[0042] Depend on image 3 It can be seen that at two different time points, Reo-3 virus infected LLC-MK2 cells, and at different dilution factors, all showed a good linear relationship, and the results were stable and reproducible.

Embodiment 4

[0043] Comparison of different experimental operators in embodiment four

[0044] According to the operation process of Example 1, two experimental operators, Operator 1 and Operator 2, independently carried out experiments at the same time, Run1 and Run2, each Run contained 6 samples of experiments, each sample was repeated three times, and the samples were serially diluted , do 8 duplicate hole experiments for each dilution, exclude the samples whose virus concentration is lower than the detection limit, select four of them for result analysis, the results are as follows Figure 4 shown.

[0045] Depend on Figure 4 It can be seen that there is no significant difference between Operator 1 and Operator 2, and both operators show a good linear relationship under different dilution factors.

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Abstract

The invention discloses a method for staining and determining the titer of reovirus type 3 by a tissue half infection method, which comprises the following steps: by taking rhesus monkey kidney cells, namely LLC-MK2 cells, as indicator cells for detecting the reovirus type 3, namely Reo-3 virus, titrating the reovirus type 3 by the tissue half infection method; and judging a cytopathic effect by combining a crystal violet staining method, and finally calculating to obtain the titer of the virus. According to the determination method provided by the invention, the cytopathic effect is judged by combining a tissue half infection method with a crystal violet staining method, the cell condition can be simply and intuitively observed, the subjective influence of microscopic observation of experimenters is effectively reduced, meanwhile, the method is good in stability, the experiment result cannot be influenced by different time and different operators, and the method is suitable for popularization and application. And the repeatability is good.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for measuring the titer of reovirus type 3 by staining with a tissue half infection method. Background technique [0002] With the development of biotechnology, many biological drugs have entered the drug market. In addition to blood products, biological products such as monoclonal antibodies and recombinant proteins have also become biomedical products due to their strong targeting, small side effects and significant curative effect. important direction of industry development. These biological products are usually derived from organisms such as microorganisms, cells, animal or human tissues and body fluids. If animal cells are used as protein expression production systems, biological products may have the risk of virus contamination in multiple links. Due to biological Products are usually administered by direct injection, and if the product is contaminated by vir...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/02G01N1/30G01N31/16
CPCC12Q1/06G01N1/30G01N31/16G01N33/5005
Inventor 秦冲梁艳王素李童陈源源汪景长童涌
Owner SUZHOU YAOMING KANGDE INSPECTION TESTING
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