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Method for determining content of Newcastle disease virus by using full-suspension cells

A technology of chicken Newcastle disease virus and detection method, which is applied in the field of determination of chicken Newcastle disease virus content by using whole suspension cells, can solve the problems of long time-consuming, high cost of SPF chicken embryos, achieve short culture time, high sensitivity, and avoid missed detection.

Pending Publication Date: 2021-02-12
成都史纪生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the main measure to prevent and control Newcastle disease is vaccination, which is mainly divided into two categories: live vaccines and inactivated vaccines. The most important thing to control the quality of these two vaccines is that the virus content of the antigen solution used for vaccine production must meet the standard requirements. At present There is only one chicken embryo detection method used to detect the virus content of Newcastle disease virus in chickens. Since SPF chicken embryos need to be ordered in advance and can only be used after hatching to 10 days old, it takes a long time and the cost of SPF chicken embryos is high, so it is necessary to Looking for a more convenient, simple and accurate method

Method used

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  • Method for determining content of Newcastle disease virus by using full-suspension cells
  • Method for determining content of Newcastle disease virus by using full-suspension cells
  • Method for determining content of Newcastle disease virus by using full-suspension cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 Virus content detection method of the present invention

[0026] 1. Recovery and culture of fully suspended duck embryonic retinal cells (referred to as "AGE1 cells")

[0027] Take 1 cell from the liquid nitrogen tank, melt it quickly in a water bath at 37°C, centrifuge at 200g for 5 minutes, discard the supernatant, suspend the cells with 30ml of cell growth medium, inoculate them in a 125ml cell culture shaker flask, and set the temperature at 160r / min, 37°C, 5%CO 2 Cultured under the conditions, making it stably passaged for 3 times (microscopic observation results are as follows: figure 1 shown).

[0028] 2. Virus content (TCID 50 ) detection method

[0029] Get chicken Newcastle disease virus aSG10 strain (HA titer is 8log2), carry out 10-fold serial dilution, each dilution is inoculated 5 tubes of AGE1 cells, the volume of cell culture is 10ml / tube, and the inoculated cell density is 8.0×10 6 / ml, each tube of cells before virus inoculation was s...

experiment example 1

[0032] The relation of experimental example 1 culture time and sensitivity

[0033] 1. Method

[0034] Use the method of Example 1 to resuscitate AGE1 cells, pass passage to make the cell density reach 8.0×10 6 / ml, take chicken Newcastle disease virus aSG10 strain and carry out 10-fold serial dilution, take 10 -7 、10 -8 、10 -9 、10 -10 Four dilutions of virus solution were inoculated into AGE1 suspension cells, the volume of cell culture was 10ml / tube, 5 tubes were inoculated for each dilution, the inoculated volume was 0.1ml / tube, and the final concentration of 15μg was added to each tube Trypsin solution per ml, each tube of cells before inoculation was supplemented with DMEM medium accounting for 15% of the cell culture volume, 220r / min, 35°C, 5% CO 2 Cultivate under conditions, respectively, after culturing for 48h, 72h, 96h, and 120h, samples were taken for HA titer determination, and if the HA titer was not lower than 7log2, it was determined to be an infection.

...

experiment example 2

[0040] Experimental Example 2 Sensitivity comparison between the present invention and traditional SPF chicken embryo detection method (chicken embryo detection method)

[0041] 1. Method

[0042] 1) Chicken embryo detection method

[0043] Take chicken Newcastle disease virus aSG10 strain, carry out 10-fold serial dilution, take 10 -7 、10 -8 、10 -9 、10 -10 Four dilutions of the virus were inoculated with 5 10-day-old SPF chicken embryos at each dilution, 0.1ml / piece, cultured at 37°C for 120 hours, and samples were taken for HA titer determination. If the HA titer was not less than 7log2, it was determined to be an infection.

[0044] 2) method of the present invention

[0045] Same as Experimental Example 1, wherein the culture time is 72h.

[0046] 2. Results

[0047] 10 -7 and 10 -9 The infection rates of SPF chicken embryos and AGE1 cells inoculated with two dilutions were the same, and the dilution was 10 -8 and 10 -10 When AGE1 cells were inoculated, the viru...

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Abstract

The invention provides a method for determining content of Newcastle disease virus by using full-suspension cells, and belongs to the field of virus detection. The method mainly comprises the following steps: 1) diluting the newcastle disease virus into different dilutions; 2) inoculating into full-suspension duck embryo retina cells for culturing; and 3) determining HA titers with different dilutions and calculating virus titer; wherein in the step 2), pancreatin needs to be added during virus inoculation; wherein the final concentration of pancreatin is 10-20 [mu]g / ml. Through comparison, the method disclosed by the invention can be used for detecting the Newcastle disease virus in a relatively short time, and is superior to a traditional chick embryo detection method.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a method for measuring chicken Newcastle disease virus content by using whole suspension cells. Background technique [0002] Chicken Newcastle disease (ND), also known as Asian chicken plague or pseudo chicken plague, is an acute and highly contagious infectious disease of poultry caused by Newcastle disease virus (NDV). The World Health Organization (OIE) lists it as an animal disease that must be reported, and my country lists it as a first-class animal disease. [0003] At present, the main measure to prevent and control Newcastle disease is vaccination, which is mainly divided into two categories: live vaccines and inactivated vaccines. The most important thing to control the quality of these two vaccines is that the virus content of the antigen solution used for vaccine production must meet the standard requirements. At present There is only one chicken embryo det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12R1/93
CPCC12Q1/70
Inventor 丁莉刘汉平李成山左榕琳邢刚岳丰雄潘倩何洪奎肖倩裴倩如汪强
Owner 成都史纪生物制药有限公司
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