African swine fever virus ELISA antibody detection kit

An African swine fever virus and antibody detection technology, applied in measurement devices, immunoassays, instruments, etc., can solve the problems of low virus immunogenicity, missed detection, inconsistent immune sensitivity, etc., and achieve good repeatability and long shelf life. , good specificity

Pending Publication Date: 2022-02-25
ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Due to the complex infection mechanism of ASFV, there is currently no effective vaccine for prevention and treatment. In the short term, early diagnosis and regionalized prevention and control management are used for prevention and control. Strict monitoring plans are adopted. Source of infection
So far, the main diagnosis of African swine fever is based on virus gene detection, through RT-PCR and partial genome sequence analysis. In the case of low virus content, it is impossible to give an accurate judgment of infection in time. In addition, due t...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The preparation of the African swine fever virus p72, p30 and p54 protein of embodiment 1 prokaryotic expression

[0026] 1.1 Preparation of African swine fever virus p72 protein

[0027] 1.1.1 Construction of recombinant Escherichia coli engineering bacteria E.coli BL21 / pet28a-p72

[0028] The p72 protein gene sequence was sent to Huada Gene Technology Co., Ltd. for full-sequence synthesis and ligated to the pet28a plasmid. The ligated plasmid was transformed into Escherichia coli BL21 (DE3), and a single clone was picked in a medium containing 100 μg / ml kanamycin The LB medium was cultured overnight, and the plasmid was extracted and analyzed by sequencing. The positive clone was the engineering bacterium E.coli BL21 / pet28a-p72.

[0029] protein expression

[0030] Inoculate 0.5ml of E.coli BL21 / pet28a-p72 strain into 50ml of LB liquid medium containing kanamycin, shake at 220 rpm at 37°C for 4-5 hours, OD 600 To 0.6-0.8, add IPTG solution with a final concentratio...

Embodiment 2

[0042] An African swine fever virus ELISA antibody detection kit, the kit includes an enzyme-labeled plate coated with an antigen, a blocking solution, a sample diluent, a washing solution, a color developing solution, an enzyme-labeled conjugate, a stop solution, and an African swine Pestivirus-positive pig serum control and African swine fever virus-negative pig serum control, the antigens are the prokaryotic expressed African swine fever virus p72, p30 and p54 proteins prepared in Example 1.

[0043] The preparation method of the enzyme label plate that is coated with antigen comprises the following steps: the African swine fever virus p72, p30 and p54 protein of the prokaryotic expression that embodiment 1 makes with the carbonate buffer solution of pH9.6 according to mass ratio 1 After 1:1 dilution to an antigen solution with a concentration of 0.1 μg / mL, add 100 μL of the diluted antigen solution (antigen coating amount 10 ng) to each well of the microplate, seal the plat...

Embodiment 3

[0057] Example 3 Test experiments such as specificity and sensitivity of the kit

[0058] 3.1 Specificity test

[0059] The African swine fever virus ELISA antibody detection kit prepared in Example 2 is positive to swine fever virus antibody positive serum, porcine circovirus type 2 antibody positive serum, porcine reproductive and respiratory syndrome virus antibody positive serum, porcine pseudorabies virus antibody positive Serum, porcine parvovirus antibody-positive serum, and African swine fever virus antibody-positive serum samples were tested by ELISA, each sample was repeated three times, and a positive control (African swine fever virus positive pig serum control) and a negative control (African swine fever virus positive pig serum control) and negative control (African swine fever virus positive serum control) were set up Pestivirus negative pig serum control).

[0060] The test results show that, except that the African swine fever virus clinical antibody positive...

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Abstract

The invention discloses an African swine fever virus ELISA antibody detection kit, and belongs to the technical field of virus detection, the kit comprises an ELISA plate coated with antigens, and the antigens are prokaryotically expressed African swine fever virus p72, p30 and p54 proteins. The ELISA plate of the kit is coated with antigens, the antigens are prokaryotically expressed African swine fever virus p72, p30 and p54 proteins, the antigens are coated with extremely low antigen content, and the kit has no cross reaction with other main swine diseases and has good specificity through a specific test; the kit is high in sensitivity, good in repeatability and long in storage life, antibodies in different periods can be effectively detected through combined use of the three kinds of protein p72, p30 and p54 of the African swine fever viruses, a basis is provided for clinical swine herd infection conditions, and the kit plays an important role in preventing and controlling African swine fever virus infection.

Description

technical field [0001] The invention belongs to the technical field of virus detection. Background technique [0002] African swine fever (ASF) is an acute, febrile and highly lethal infectious disease of pigs caused by African swine fever virus (ASFV). It is characterized by a short course of disease and a mortality rate as high as 100%. The clinical symptoms and pathological changes are similar to those of acute swine fever, with high fever, skin congestion and cyanosis, miscarriage, edema, and organ bleeding. [0003] ASF first appeared in Kenya in 1921, and became popular in Africa, Europe, South America and other countries in the 1950s and 1960s. In recent years, ASF has spread to Georgia, Armenia, Azerbaijan, Russian Federation and other neighboring countries of our country, and there is a tendency of continuous spread, which poses a serious threat to my country's pig industry. [0004] African swine fever virus is the only member of the ASF-like virus family. The di...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543
CPCG01N33/56983G01N33/54306G01N2333/01G01N2469/20
Inventor 吴海龙赵林萍付燕峰曾小宇李慧英娄亚坤常伟任宝红
Owner ZHENGZHOU ZHONGDAO BIOTECHNOLOGY CO LTD
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