IPMA neutralizing antibody detection method for PRRSV
An antibody detection and cell technology, applied in the biological field, can solve the problems of inability to evaluate PRRSV-specific antibody response and development, and achieve high sensitivity, simple operation, and strong specificity
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Embodiment 1
[0024] Example 1 PRRSV isolation and identification
[0025] Rinse the suspected PRRSV disease tissue (lymph, liver, spleen, lung) with PBS, cut it into pieces, add 1mL PBS to 1g of the tissue sample, freeze and thaw three times, and make the disease material grinding liquid (used as PRRSV virus liquid), Use the RNA extraction kit to extract the RNA of PRRSV from the above disease materials, and then reverse transcribe the RNA into cDNA.
[0026] Use DNAMAN software to design PRRSV target gene primers, and the specific primer sequences are as follows:
[0027] PRRSV-F: 5'-GGCCAGCCAGTCAATCAG-3'; SEQ ID NO.1;
[0028] PRRSV-R: 5'-GGCAAACTAAACTCCACAGTG-3'; SEQ ID NO.2.
[0029] Utilizing the designed primers, the above-mentioned extracted PRRSV lymph, liver, spleen, and lung cDNAs were used as templates for PCR amplification. The PCR amplification system is 25 μL: template 1 μL, PRRSV-F 0.5 μL, PRRSV-R 0.5 μL, Ex Taq enzyme 12.5 μL, double distilled water 10.5 μL. The reactio...
Embodiment 2
[0030] Example 2 establishes a stable IPMA neutralizing antibody detection method
[0031] Marc145 cells were digested and centrifuged to prepare 1.0×10 5 ceLs / 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 PRRSV virus (50 μL) and clinical serum at 37°C for 1 hour, and then inoculate them on Marc145 cells in a 96-well plate. After culturing for 48 hours, take out 96 cells after the cells are full. The well plate was fixed with pre-cooled absolute ethanol. Add PRRSV standard positive serum, incubate at 37°C for 1h, then add goat anti-pig IgG-HRP, incubate at 37°C for 1h, develop color with DAB or AEC, observe under an ordinary optical microscope, if a reddish-brown insoluble product is produced, it is negative, otherwise it is Positive. The serum identified as positive was serially diluted, and the above operati...
Embodiment 3
[0038] Embodiment 3 IPMA reaction conditions
[0039] 1) TCID of PRRSV 50 determination
[0040] (1) Spread the Marc145 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;
[0041] (2) In the penicillin bottle or the centrifuge tube, the PRRSV virus liquid is diluted 10 times continuously, starting from 10 -1 -10 -10 ;
[0042] (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;
[0043] (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);
[0044] (5) After culturing for 48 hours, the cells were taken out and fixed, and placed at -20°C for later us...
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