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Quantitative detection method of goat pox virus

A quantitative detection method and technology for goat pox virus, which can be used in measurement devices, resistance to vector-borne diseases, instruments, etc., can solve the problems of test failure, cumbersome operation, long time consumption, etc., and achieve short detection time, good repeatability, and improved quality effect

Active Publication Date: 2022-05-27
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above two methods are all based on nucleic acid detection methods, need to design primers, and primer synthesis, and CN104152583B needs fluorescent dyes, environmental friendliness needs to be improved, and can only determine whether there is a goat pox virus gene sequence in the qualitative sample, and cannot Intact virus for accurate quantification
[0004] TCID 50 The method is cumbersome and time-consuming. The virus needs to be mixed with cells in multiple dilutions and placed in a carbon dioxide incubator for 7 days. The TCID of the virus is determined according to the number of cytopathic changes. 50 (half the amount of cell infection)
This method has high requirements on the aseptic conditions of the environment. If the operation is not done properly, it is easy to cause bacterial contamination and cause the test to fail.

Method used

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  • Quantitative detection method of goat pox virus
  • Quantitative detection method of goat pox virus
  • Quantitative detection method of goat pox virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The purpose of this example is to study the influence of the buffer system on the detection results.

[0048] Choose three buffers of different concentrations, namely 10mM, 20mM, 30mM phosphate buffer, add methanol with a volume ratio of 5%, 10%, and 15% respectively, and use the degree of separation as a parameter to investigate the influence of different buffer systems on the detection .

[0049] Taking the goat pox virus suspension culture medium as the research object, the influence of different phosphate buffer systems on the separation degree was investigated. The results are shown in Table 1.

[0050] Table 1. Results of the effect of buffer composition on resolution

[0051]

[0052] It can be seen from Table 1 that the best resolution is obtained by adding 10% methanol by volume to 10 mM phosphate buffer, while the resolution of other buffers is lower.

Embodiment 2

[0054] A standard curve for antigen quantification was established.

[0055] (1) Preparation of standard products:

[0056] Chromatographic detection and component collection: Take the suspension culture of goat pox virus AV41 strain, centrifuge at 3000-5000 r / min for 5-10 minutes, take the supernatant, filter it with a 0.45 μm membrane, and concentrate it 10 times with a 100KD ultrafiltration membrane. Detected by liquid chromatograph, the absorption spectrum at 280nm was obtained. Collect the components.

[0057] Virus identification: The collected components were specifically identified by a virus-specific detection method. The method is as follows: each component is mixed with an equal amount of MEM nutrient solution, and at the same time, it is treated with blank control (cell growth solution) at 37° C. for 1 hour. Inoculate 6-well cell culture plates that have grown into a well-grown MDBK cell monolayer, 1 ml per well, and 4 wells in each group. Set at 37°C, with 5% ...

Embodiment 3

[0071] Quantitative detection of goat pox virus in suspension culture

[0072] 1. Instruments and reagents

[0073] Liquid chromatograph AKTA pure with UV detector. Protein quantifier Thermo Nanodrop2000C.

[0074] Preparation of mobile phase: take 0.2M Na 2 HPO4 aqueous solution 72ml and 0.2M NaH 2 28 ml of PO4 aqueous solution, dilute to 2000 ml with ultrapure water, prepare a buffer solution containing 10 mM phosphate, add 10% methanol by volume, pH 7.2 to obtain a mobile phase, which is filtered with a 0.45 μm membrane.

[0075] Sample to be tested: goat pox virus suspension culture (AV41 strain, provided by China Animal Husbandry Industry Co., Ltd.)

[0076] Standard product: prepared by China Animal Husbandry Industry Co., Ltd., from Example 2.

[0077] 2. Determination of goat pox virus content in suspension culture medium

[0078] The goat pox virus cultured in suspension was quantified, the samples were clarified by centrifugation (the samples to be tested were ...

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Abstract

The invention discloses a quantitative detection method of a goat pox virus. The method comprises the following steps: firstly, identifying a target peak by using a virus specificity detection method; determining the concentration of the standard substance by using a protein determinator; carrying out series dilution on the standard substance, detecting the absorption peak of the standard substance at 280nm on a liquid chromatograph, and establishing a linear regression equation of concentration and peak area; and calculating the content of the goat pox virus in the sample to be detected according to the linear regression equation and the peak area of the sample to be detected. The method disclosed by the invention can be used for determining the content of the goat pox virus in a goat pox virus suspension culture solution, a purified solution and a goat pox inactivated vaccine, has the advantages of rapidness, accuracy, stability and good repeatability, can guide the production of vaccines, and plays an important role in improving the quality of the vaccines.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to a virus detection and quantitative method, in particular to a quantitative detection method of goat pox virus. Background technique [0002] Capripoxvirus (GPV) is a member of the Poxviridae family, and is a double-stranded DNA virus with a linear genome of about 150kb in length, encoding 147 open reading frames. The basic chemical components of viruses are proteins, DNA and lipids. The virions are brick-shaped with a size of 300nm×270nm×200nm. GPV can infect goats of all breeds, sexes and ages, with lambs being the most susceptible, with infection rates up to 100%. GPV infects other goats through contact with damaged skin, respiratory inhalation, or vector transmission. Goat pox virus has high resistance to drying, the virus in the dry crust can survive for 3 to 6 months, and it can still survive for about 5 minutes when the dry heat reaches 100 ℃. It has strong resistance to common d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/027G01N2030/042Y02A50/30
Inventor 宋芳巴利民杨君敬潘文
Owner CHINA ANIMAL HUSBANDRY IND
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