A kind of isolation method of porcine parvovirus

A parvovirus and separation method technology, applied in the direction of virus, virus/bacteriophage, single-stranded DNA virus, etc., can solve the problems of unfavorable timely diagnosis of porcine parvovirus disease, cumbersome virus sample processing process, and low virus purity, etc. Timely diagnosis and research, shortened separation cycle, simple and fast preparation

Active Publication Date: 2020-11-17
浙江美保龙生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In view of the fact that the disease is prevalent in the world and seriously endangers the pig industry, the rapid isolation and identification of porcine parvovirus plays an important role in the control of porcine parvovirus disease. However, the traditional virus isolation method takes a long time and the virus sample processing process is cumbersome. Moreover, it is easy to pollute, and the purity of the isolated virus is not high, which is not conducive to the timely diagnosis of porcine parvovirus disease. Therefore, the invention of a rapid separation and identification method for porcine parvovirus is of great significance for the rapid diagnosis and research of this disease.

Method used

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  • A kind of isolation method of porcine parvovirus
  • A kind of isolation method of porcine parvovirus
  • A kind of isolation method of porcine parvovirus

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1: Preparation of CEF primary cells

[0026] Take 9-10-day-old SPF chicken embryos with rich blood vessels and vigorous vitality. After disinfection, use large-head tweezers to gently break the air chamber of the chicken embryo body to expose the embryo body. After carefully picking out the embryo body, place it in a pH 7.0 PBS solution containing berberine (each 1000ml contains 6-10 g sodium chloride, 0.05-0.5 g potassium chloride, 1-1.2 g disodium hydrogen phosphate, 0.05-0.5 g potassium dihydrogen phosphate, 0.05-0.05 g calcium chloride 0.2 g, magnesium chloride containing 6 crystal water 0.05-0.2 g, berberine 1-3 mg; mix the above ingredients, dissolve in 1000 ml double distilled water, filter through a 0.22 um filter membrane, and place in Store at 4°C for later use; or sodium chloride 6-10 g, potassium chloride 0.05-0.5 g, disodium hydrogen phosphate 1-1.2 g, potassium dihydrogen phosphate 0.05-0.5 g, calcium chloride 0.05-0.2 g, containing 6 Magnesium ch...

Embodiment 2

[0027] Example 2 Collection and processing of disease materials

[0028] Aseptically collect the heart, liver, spleen, lung, and kidney of the stillborn and mummified fetuses of breeding sows, place them in a meat grinder and grind them, add D-Hanks solution twice the total mass of the minced meat to the minced meat, and grind through colloid Grind well and mix well. The obtained mixed solution is placed in a high-pressure homogenizer, and the temperature is controlled below 25° C. during the homogenization process to obtain a homogenization treatment solution. Add D-Hanks solution to the homogenate treatment solution at a ratio of 1:1, 1:2 or 1:3 (each 1000 ml consists of the following ingredients: 8-10g of sodium chloride, 0.4-0.6g of potassium chloride, containing 1 Disodium hydrogen phosphate 0.04-0.08g, Potassium dihydrogen phosphate 0.04-0.06g, Sodium bicarbonate 0.3-0.5g, Phenol red 0.01-0.02g; Mix the above ingredients thoroughly, dissolve in 1000ml double distilled w...

Embodiment 3

[0029]Example 3: Cell inoculation of virus

[0030] The primary CEF cells that have been cultured for 24 hours and grew well, the cell culture medium was discarded, and the virus stock solution and cell culture medium were added at a ratio of 1:8, 1:9 or 1:10 (V:V), and a positive control of PPV was set at the same time and the blank control group, and then placed at 37°C, 5% CO 2 Incubate in the incubator for 1 hour, discard the virus solution, add virus maintenance solution containing 2% fetal bovine serum, continue to cultivate for 90 hours, observe cell changes every day, negative and positive control groups are established, collect the cell solution with obvious changes, and place in - Store at 20°C.

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Abstract

The invention discloses a separation method for a porcine parvovirus and belongs to the technical field of veterinary biological products. The method comprises the following steps: (1) preparation of a CEF primary cell; (2) collection and treatment of a pathological material; (3) cell inoculation of a virus; and (4) virus identification. The separation method for the porcine parvovirus has the beneficial effects that the treatment method of the pathological material is free of freeze thawing, high in mechanical degree, time-saving and labor-saving; and a susceptible primary cell for culturing the porcine parvovirus is provided, the CEF primary cell is simple and fast in preparation and low in cost, the separation cycle of the virus is greatly shortened, and timely diagnosis and research of the porcine parvovirus are facilitated.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, in particular to a method for isolating porcine parvovirus. Background technique [0002] Porcine parvovirus (PPV) is a member of the genus Parvovirus in the family Parvoviridae and is a self-replicating virus. PPV is one of the important pathogens causing reproductive disorders in sows, which can lead to miscarriage, premature birth, stillbirth, mummified fetuses, weak piglets, infertility of sows and mass death of newborn piglets. PPV is mainly transmitted through the digestive tract and respiratory tract. The infection of boars, fattening pigs and sows is mostly due to contact with contaminated feed and drinking water. PPV can also be transmitted through the reproductive tract. Infected pigs are the main source of PPV infection. The virus in the secretions and excreta of infected pigs can remain infected for several months, and the pens contaminated by it are still infe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00
CPCC12N7/00C12N2750/14351
Inventor 沈建军张秀文李阳冷春青
Owner 浙江美保龙生物技术有限公司
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