IFA antibody detection method for PRRSV
An antibody detection and cell technology, applied in the field of IFA antibody detection of PRRSV, can solve the problems of not being developed and unable to evaluate the PRRSV-specific antibody response, etc., and achieve the effect of simple operation, high sensitivity and strong specificity
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Embodiment 1
[0027] Example 1 PRRSV isolation and identification
[0028] 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.
[0029] Use DNAMAN software to design PRRSV target gene primers, and the specific primer sequences are as follows:
[0030] PRRSV-F: 5'-GGCCAGCCAGTCAATCAG-3'; SEQ ID NO.1;
[0031] PRRSV-R: 5'-GGCAAACTAAACTCCACAGTG-3'; SEQ ID NO.2.
[0032] Using the designed primers, PCR amplification was carried out using the above-mentioned extracted PRRSV lymph, liver, spleen, and lung cDNA as templates. The system was 25 μL, and the reaction conditions were: pre-denaturation at 98°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55°C ...
Embodiment 2
[0033] Example 2 establishes a stable IFA antibody detection method
[0034] The virus liquid was inoculated into Marc145 cells and fixed with pre-cooled absolute ethanol (it can be prepared in advance and stored for later use). Add PRRSV standard positive serum, incubate at 37°C for 1.0h, then add goat anti-pig IgG-FITC, incubate at 37°C for 1h, observe under a fluorescent microscope, if specific green fluorescence is produced, it is positive, otherwise it is negative. The serum identified as positive was serially diluted, and the above operation was repeated to obtain the antibody titer.
[0035] (1) Inoculation: when the Marc145 cells in the cell bottle grow to about 50-70%, 100 TCID 50 The PRRSV virus solution was inoculated onto Marc145 cells, and cultured by adding DMEM medium containing 2% fetal bovine serum;
[0036] (2) Plating: After the cells are overgrown, spread them into a 96-well plate, inoculate 100 μl in each well, and place in CO 2 Continue culturing in th...
Embodiment 3I
[0041] Embodiment 3IFA reaction conditions
[0042] 1) TCID of PRRSV 50 determination
[0043] (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;
[0044] (2) In the penicillin bottle or the centrifuge tube, the PRRSV virus liquid is diluted 10 times continuously, starting from 10 -1 -10 -10 ;
[0045] (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;
[0046] (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);
[0047] (5) After culturing for 48 hours, the cells were taken out and fixed, and placed at -20°C for later use;...
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