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IFA antibody detection method of CSFV

An antibody detection and cell technology, which is applied in the field of IFA antibody detection of CSFV, can solve the difficulties in the diagnosis and prevention of classical swine fever, troubled the pig industry and other problems, and achieve the effect of 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

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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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  • IFA antibody detection method of CSFV
  • IFA antibody detection method of CSFV
  • IFA antibody detection method of CSFV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 CSFV Virus Isolation and Identification

[0023] 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:

[0024] CSFV-F: 5'-cggctagcctgcaaggaagattac-3'; SEQ ID NO.1;

[0025] CSFV-R: 5'-tcatagatcttcattttccactgtggtgg-3'; SEQ ID NO.2.

[0026] 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

[0028] The establishment of embodiment 2 clinical antibody detection method

[0029] Normal PK15 cells were divided into 1.0×10 5 Inoculate to 96-well cell culture plate, after the cells adhere to the wall, the CSFV virus liquid isolated in Example 1 is diluted 1:10 with DMEM containing 2% fetal bovine serum, and then added to the 96-well cell culture plate, each well 100 μL, and set up a negative control at the same time. Put in 37℃5%CO 2 Culture in an incubator for 48 hours, and observe the cell growth every day.

[0030] After 2 days, discard the medium in the 96-well cell culture plate, wash the cells gently with PBST 3 times, and pat dry; add pre-cooled absolute ethanol, 50 μL per well, place at -20°C for 30 minutes, take it out, and discard the absolute ethanol , washed 5 times with PBST, and patted dry; added 5% skimmed milk powder to block, 200 μL per well, and placed in a 37°C incubator for 2 hours; washed 5 times with PBST, patted dry; added CSFV positive antibody...

Embodiment 3

[0032] Embodiment 3 IFA reaction conditions

[0033] 1) CSFV TCID 50 determination

[0034] (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;

[0035] (2) In the penicillin bottle or the centrifuge tube, the CSFV virus liquid is diluted 10 times continuously, starting from 10 -1 -10 -10 ;

[0036] (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;

[0037](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);

[0038] (5) After culturing for 48 hours, the cells were taken out and fixed, and placed at -20°C for later use;

[003...

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Abstract

The invention discloses an IFA antibody detection method of CSFV and belongs to the technical field of biology. According to the IFA antibody detection method of the CSFV, the cell reaction plate can be prepared in advance and can be stored for a long time at 20 DEG C; reaction results can be observed by a fluorescence microscope. The method has the characteristics of strong specificity, high sensitivity, simple operation and long-term preservation. Whole viruses are used for antibody detection, and the antibody level of animals can be truly reflected.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a CSFV IFA 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). For high fever missed 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 of the family Flaviviridae, is...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/58
CPCG01N33/56983G01N33/582G01N2333/183G01N2469/20
Inventor 李鹏王利平刘兴友金前跃王寅彪陈磊山刘长忠孙国鹏岳锋李红张艳芳齐永华潘鹏涛
Owner XINXIANG UNIV
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