Construction and application of Marek's disease virus ultra-high virulent strain and gene deletion virulent strain of Marek's disease virus ultra-high virulent strain
A technique for Marek's disease, a super-virulent strain, applied in the field of animal virology
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Embodiment 1
[0042] Example 1: MD clinical sample collection and virus isolation and culture
[0043] A flock of 120-day-old Hailan brown laying hens in a farm in Luohe City, Henan Province became ill. The clinical manifestations of the sick chickens were sluggishness, necking, fluffy feathers, and loose yellow stools. Most of the chickens were stunted and extremely thin. Neurological symptoms such as limb splits, twisted neck and crooked neck. The incidence of chicken flocks is as high as 35%, and the mortality rate is about 15%. The necropsy of the sick and dead chickens showed that most of the sick and dead chickens showed hepatosplenomegaly, and some chickens had one or more focal tumors or diffuse tumor nodules in internal organs, which were clinically diagnosed as suspected cases of MD.
[0044] Seven live chickens from the above-mentioned suspected cases were taken, numbered 1 to 7 in sequence, and 2 to 3 mL of blood was collected from the subwing vein aseptically, and heparin sodi...
Embodiment 2
[0045] Example 2: Cloning, purification and identification of MDV epidemic strain HN302
[0046] 1. PCR screening and identification of cell cultures
[0047] For the conserved region of the MDV-1 specific tumorigenic gene meq sequence, a primer pair for PCR amplification of the meq gene was designed and synthesized: meq-F 5'-ATGTCTCAGGAGCCAGAG-3' (SEQ ID NO. 2) and meq-R 5 '-TCAGGGTCTCCCGTCA-3' (SEQ ID NO. 3), and then PCR identification was performed on the DNA sample of the second-generation virus isolation cell culture prepared in Example 1.
[0048] The PCR reaction system included: 10 μL of 2×EasyTaq PCR SuperMix (+dye), 0.5 μL of 10 μM upstream and downstream primers, 1 μL of 10 ng / μL DNA template of the sample to be tested, and 7.5 μL of sterilized ultrapure water.
[0049] The PCR reaction program was as follows: pre-denaturation at 94°C for 4 min; 30 cycles of denaturation at 94°C for 30s, annealing at 58°C for 30s, extension at 72°C for 1 min; extension at 72°C for...
Embodiment 3
[0060] Example 3: Pathogenicity Analysis and Pathotype Identification of HN302
[0061] 1. Determination of viral titer of HN302
[0062] Resuscitate the HN302 virus that was frozen in liquid nitrogen, dissolve rapidly in a 37°C water bath, and centrifuge at 1,000 r / min for 5 minutes, discard the cell cryopreservation solution, and resuspend the cells in 1 mL of M199 medium containing 2% (v / v) FBS. After mixing well, 900 μL was aspirated, and the HN302 virus solution was mixed with M199 medium containing 2% (v / v) FBS on a 24-well plate according to 2 -1 ~2 -12 Make a doubling dilution; then 2-6 ~2 -12 The diluted virus solution was resuspended and transferred to CEF monolayer cells in a 48-well plate, 300 μL / well, and each dilution was repeated 3 times. The last column of cell wells was directly supplemented with 2% (v / v) FBS. M199 medium was used as a negative control at 38.5°C, 5% CO. 2 After culturing in an incubator for 3-4 days, after typical virus plaques were observ...
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