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IPMA neutralizing antibody detection method of CSFV

An antibody detection and cell technology, which is applied in the field of CSFV IPMA neutralizing antibody detection, can solve the problems that plague the pig industry, the diagnosis and prevention of swine fever, and achieve high sensitivity, simple operation, and strong specificity

Pending Publication Date: 2021-04-06
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These vaccines have been able to control the prevalence of swine fever very well, but due to the long-term use of the vaccine, the prevalence of swine fever has undergone major changes, mainly becoming atypical swine fever and mild swine fever
This has caused difficulties in the diagnosis and prevention of swine fever, and it has also made swine fever prevalent, which has plagued China's pig industry

Method used

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  • IPMA neutralizing antibody detection method of CSFV
  • IPMA neutralizing antibody detection method of CSFV
  • IPMA neutralizing antibody detection method of CSFV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1CSFV virus isolation and identification

[0025] After washing the suspected CSFV disease tissue (lymph, liver, spleen, lung) with PBS for several times, add 1 mL of PBS to dissolve 1 mg of tissue sample, freeze and thaw repeatedly 3 times, take the supernatant, and then centrifuge at 12000 rpm for 5 min, and use 0.22 Filter with a μm filter to obtain CSFV virus liquid. Using a DNA extraction kit, extract the RNA of CSFV virus, and then reverse transcribe the RNA into cDNA. Use DNAMAN software to design primers for the target gene of CSFV, the amplified fragment is 531bp, and the specific primer sequences are as follows:

[0026] CSFV-F: 5'-cggctagcctgcaaggaagattac-3'; SEQ ID NO.1;

[0027] CSFV-R: 5'-tcatagatcttcattttccactgtggtgg-3'; SEQ ID NO.2.

[0028] Using the designed primers, PCR amplification was performed using the extracted cDNA as a template. The reaction system was as follows: template 1 μL, CSFV-F 0.5 μL, CSFV-R 0.5 μL, Ex Taq enzyme 12.5 μL...

Embodiment 2

[0030] Example 2 Establishment of Clinical Serum Neutralizing Antibody Detection Method

[0031] Digest and centrifuge PK15 cells to prepare 1.0×10 5 cells / mL cell suspension, spread 100 μL per well on a 96-well plate, and store at 37°C in 5% CO 2 Cultivate in the incubator for 12 hours. After the cells are completely adhered to the wall, incubate an equal volume of CSFV virus (50 μL) and clinical serum at 37°C for 1 hour, and then inoculate them on PK15 cells in a 96-well plate; at the same time, set up a control well for normal inoculated cells; After culturing for 48 hours, the 96-well plate was taken out after the cells were overgrown, and fixed with pre-cooled absolute ethanol. Add primary antibody: Wash the 96-well plate 2-3 times with PBS, then add 100 μL of CSFV-positive serum diluted 1:400 with 0.01M PBS (according to the detection method of IDEXX kit, CSFV-positive antibody obtained clinically), set Act at 37°C for 1h. Add secondary antibody: add 100 μL of goat an...

Embodiment 3I

[0033] Embodiment 3IPMA reaction conditions

[0034] 1) CSFV TCID 50 determination

[0035] (1) Spread the PK15 cell suspension on a 96-well plate, 100 μL per well, so that the cell volume reaches 2-3×10 5 cells / mL, cultivated for 12 hours until the cells were completely attached to the wall;

[0036] (2) In the penicillin bottle or the centrifuge tube, the CSFV virus liquid is diluted 10 times continuously, starting from 10 -1 -10 -10 ;

[0037](3) Inoculate the diluted virus onto a 96-well plate in which the cells grow into a single layer, inoculate a vertical row of 8 wells for each dilution, and inoculate 100 μL in each well;

[0038] (4) The remaining two vertical rows are not exposed to poison, and a normal cell control is set (100 μL of maintenance solution per well, and the maintenance solution is DMEM medium containing 2% fetal bovine serum);

[0039] (5) After culturing for 48 hours, the cells were taken out and fixed, and placed at -20°C for later use;

[004...

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Abstract

The invention discloses an IPMA neutralizing antibody detection method of CSFV and belongs to the technical field of biology. The invention discloses an IPMA neutralizing antibody detection method for CSFV. The IPMA neutralizing antibody detection method comprises steps of virus inoculation, plate laying, fixation, primary antibody addition, secondary antibody addition and color development. The method provided by the invention provides certain technical support for prevention, vaccine evaluation and immune evaluation of pigs infected with CSFV; and the serum neutralization test can be used for detecting the level of the CSFV antibody and evaluating the immune effect of the CSF vaccine, and is expected to be applied to the production of actual vaccines. According to the method, target cells are infected after interaction of whole viruses and antibodies, and the situation of interaction of viruses and neutralizing antibodies is truly reflected; the kit has the characteristics of strong specificity, high sensitivity and simple operation.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a CSFV IPMA neutralizing antibody detection method. Background technique [0002] Classical Swine Fever (Classical Swine Fever), earlier known as Hog Cholera (Hog Cholera, HC), is an acute, febrile and highly contagious infectious disease of pigs caused by Classical Swine Fever Virus (CSFV), characterized by Excessive high fever and generalized hemorrhage. The morbidity and mortality of swine fever are very high, mainly divided into acute type, subacute type, chronic type, atypical swine fever or mild swine fever, causing huge economic losses to the swine industry all over the world. It is popular all over the world to varying degrees. my country lists this disease as a first-class animal disease, and the World Organization for Animal Health (OIE) lists it as an animal disease that must be reported. Classical swine fever virus (CSFV) belongs to the genus Pestivirus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/183G01N2469/20
Inventor 李鹏王利平刘兴友王选年刘长忠魏小兵孙国鹏岳锋李红张艳芳
Owner XINXIANG UNIV
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