Immunofluorescent reagent used for detecting Muscovy duck gosling blast diseases
An immunofluorescence and goose blast technology, applied in fluorescence/phosphorescence, measuring devices, instruments, etc., can solve the problems of lack of rapid diagnostic reagents and difficulties in clinical diagnosis of muscovy duck and goose plague, and achieve innovation, practicability, and accuracy High-performance, short-term effect
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[0020] 3.1 Preparation of GPV-infected cells: Muscovy duck gosling plague virus (GPV) was inoculated into a 96-well MDEF cell culture plate that had grown into a monolayer, and normal MDEF cells were set as a negative control; cultured at 37°C under 5% CO2 conditions When the cytopathic effect reaches about 30%, discard the culture supernatant, wash once with sterile PBS or normal saline, add 100 μL of cold methanol to each well, fix at 4°C for 30 min, discard the fixative and wash once as before, -40 Store at ℃ for later use.
[0021] 3.2 Preparation of tissue frozen sections: Take the liver, spleen, pancreas and other tissues of the diseased Muscovy duck artificially infected with gosling plague virus (GPV) or the embryonic heart of the dead Muscovy duck inoculated with GPV, and cut into 0.5 cm 3 After the tissue is frozen, it is cut into 6-8um thin slices, adhered to the glass slide, fixed in cold acetone for 10-15 minutes, and frozen at -20°C for later use.
[0022] 3.3DF...
experiment example
[0036] According to the direct immunofluorescence assay (DFA) operation method, after the fluorescent antibody is appropriately diluted, the infected cells such as GPV, MPV, MDRV, ARV, PMV and normal control cells are respectively taken, and 30ul of 1:150 diluted fluorescent reagent is added to each well. DFA test. The results showed that weak positive fluorescence could be seen in the nucleus at the initial stage of GPV infection of MDEF cells, and a strong bright green fluorescence appeared in the nucleus 36 hours after infection, and then the fluorescence gradually appeared in the whole cell and went extracellular with the cell disintegration. However, other virus-infected cells and normal MDEF cells showed no fluorescence. It shows that GPV immunofluorescence reagent can be used for the identification and antigen localization of GPV-infected cells.
[0037] Table 2. Identification of GPV antigens by immunofluorescence assay (DFA)
[0038]
[0039] Note: Judgment crite...
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