Application of flow cytometry method to vaccine production real-time monitoring

A technology for flow cytometry and vaccine production, which is applied in the application field of flow cytometry method in real-time monitoring of vaccine production, can solve the problem of high sensitivity of culture method, inability to achieve instant control of the fermentation process of bacterial liquid, inability to distinguish between live bacteria and Dead bacteria and other problems, to achieve the effect of precise quality control and reliable bacterial activity monitoring methods

Active Publication Date: 2014-12-10
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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Problems solved by technology

The culture method has high sensitivity but takes a long time, and it usually takes more than 24 hours to obtain the result, which cannot achieve the purpose of immediate control of the fermentation process of the bacterial liquid; the turbidimetric method is simple to operate but uses naked eyes to observe, the detection sensitivity is poor, and it cannot distinguish between live bacteria and dead bacteria

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  • Application of flow cytometry method to vaccine production real-time monitoring
  • Application of flow cytometry method to vaccine production real-time monitoring
  • Application of flow cytometry method to vaccine production real-time monitoring

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] (1) Establish the blank control and fluorescence intensity compensation control of Mycoplasma gallisepticum cells

[0061] ① Activate Mycoplasma gallisepticum F-36 strain (China Veterinary Drug Administration) with modified Frey's medium, culture at 37°C for more than 80 hours, inoculate shake flask fermentation medium (improved Frey's medium), and culture at 37°C until bacterial growth In the logarithmic phase, take 2 tubes of fermentation broth, 1 mL each; centrifuge the fermentation broth (9000 rpm for 10 min), discard the supernatant, and obtain bacterial cells. Add 1 mL of ethanol solution with a mass volume ratio of 70% to one of the tubes of bacterial cells, mix well and let it stand for 1 hour, or add 1 mL of PBS, and boil for 10 minutes, the purpose is to inactivate and obtain the dead bacteria control; Treated bacterial cells were used as viable control.

[0062] ② Wash the two kinds of cells in step ① twice with PBS (pH7.4, 0.01mol / L), and then resuspend the...

Embodiment 2

[0078] (1) Establish the fluorescence intensity blank control and compensation control of Clostridium welchii cells

[0079]① Clostridium welchii (ATCC13124, purchased from American type culture collection) was inoculated with liquid thioglycollate (FT) medium for seed medium, activated at 37°C for 24 hours, and inoculated with shake flask fermentation medium (FT culture medium). base), cultured at 37°C for 24 hours to the logarithmic phase of bacterial growth, took 2 tubes of fermentation broth, 1 mL each; centrifuged the fermentation broth (5000 rpm for 10 min), discarded the supernatant, and obtained bacterial cells. Add 1mL PBS (pH6.2, 0.01mol / L) to one tube of bacterial cells, boil for 5min, the purpose is to inactivate and obtain dead bacteria control; at the same time, use the other tube of untreated bacterial cells as a live bacteria control .

[0080] ②Wash the two kinds of cells in step ① twice with PBS, and then resuspend to OD with PBS 600 is 0.004.

[0081] A. ...

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Abstract

The invention discloses the application of a flow cytometry method to vaccine production real-time monitoring. The application is to apply the flow cytometry method to the vaccine production process, and monitor the viable bacteria count in real time during the vaccine production process. The application comprises the steps of dyeing bacterial cell through fluorescent dye, detecting the change of bacteria fluorescence signals through a flow cytometer, and obtaining an FCM diagram with at least two types of the following parameters by measuring the change of the bacteria fluorescence signals: viable bacteria count, dead bacteria count and total bacteria count, obtaining the ratio of the viable bacteria count according to the FCM diagram, obtaining the concentration of target bacteria solution through an absolute counting method, and obtaining the viable bacteria amount and the dead bacteria amount of target bacteria according to the ratio of the viable bacteria count and the concentration of the target bacteria solution. The invention provides a convenient, rapid and reliable bacterial activity monitoring method for the vaccine production process and is convenient and easy, the operation time is only 30 minutes, and the quality of the vaccine production process can be accurately controlled.

Description

technical field [0001] The invention relates to the technical application of flow cytometry, in particular to the application of the flow cytometry method in real-time monitoring of vaccine production. Background technique [0002] The control of the bacterial fermentation process in the vaccine production process is a key link in vaccine production. Various factors such as temperature, pH value, and nutritional status of the culture medium all affect the fermentation process of the bacterial solution. Therefore, the establishment of a simple, rapid and reliable detection method for bacterial activity has practical significance for the monitoring of vaccine production. Traditional methods for monitoring vaccine production mainly include culture counting and turbidimetric counting. At present, vaccine manufacturers still rely on these two methods for vaccine quality control. The culture method has high sensitivity but takes a long time. It usually takes more than 24 hours t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
Inventor 孙俊颖刘志成郭鹏举
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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