Method for separating new type duck reovirus from hybrid virus sample
A technology for duck reovirus and virus, which is applied in the field of isolation of new duck reovirus, and can solve the problems of difficulty in separating new duck reovirus and Muscovy duck reovirus vaccine strains.
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Embodiment 1
[0029] A method for isolating novel duck reovirus from mixed virus samples, comprising the following steps:
[0030] 1) Prepare a continuous density gradient sucrose solution: from top to bottom, use equal volumes of sucrose solutions with concentrations of 20%, 30%, 40% and 50% in sequence, and obtain a continuous density gradient sucrose solution at 4°C overnight;
[0031] 2) Preparation of mixed virus liquid: the mixed virus of the new duck reovirus and the Muscovy duck reovirus vaccine strain was isolated from the disease material by conventional methods, and the above mixed virus was expanded and cultivated by cell culture to obtain the mixed virus liquid ;
[0032] 3) Prepare the virus suspension: add the mixed virus solution into the concentrating tube, then add about 4-5mL of 20% sucrose solution at the bottom of the tube, preferably to cover the bottom of the tube; ultra-fast freezing at 40,000rpm, 4°C Centrifuge for 60 minutes, concentrate and dissolve the precipita...
Embodiment 2
[0034] Embodiment 2 RT-PCR identification
[0035] The samples obtained after density gradient centrifugation in Example 1 were numbered 1-28 from top to bottom, and RT-PCR was performed to detect whether the samples were a single new type of duck reovirus.
[0036] 2.1 RNA template preparation
[0037] Use AxyPrep Body Fluid Viral DNA / RNA Mini Kit (Cat No.: AP-MN-BF-VNA-250) to extract template RNA and store at -70°C.
[0038] 2.2PCR identification primers
[0039] Novel duck reovirus-specific primers:
[0040] F1 (SEQ ID No. 1): 5'-ACCTCAGGATATCGCTGAAACT-3'
[0041] R1 (SEQ ID No. 2): 5'-CTCCATCCCTGCAGCACATGTAAAG-3'
[0042] Muscovy duck reovirus strain-specific primers:
[0043] F2 (SEQ ID No. 3): 5'-TCGGACCTCGATGTGTGGTGGT-3'
[0044]R2 (SEQ ID No. 4): 5'-TTCGAGACCGACCGCACAGGTGAAT-3'
[0045] 2.3 PCR reaction
[0046] Reaction system: TaKaRa PrimeScript One Step RT-PCR Kit Ver.2 (TaKaRa Code: DRR055A) PCR kit was used, and the reaction system was 25 μL, as shown in ...
Embodiment 3
[0052] Embodiment 3 Purification of novel duck reovirus
[0053] Take two single samples of new duck reovirus, numbered X and Z respectively, filter and inoculate muscovy duck embryo fibroblasts, culture continuously for 3 generations, pass new duck reovirus-specific primers and Muscovy duck reovirus Vaccine strain-specific primers were again identified by RT-PCR. Depend on figure 2 It can be seen that the X and Z2 samples are all single novel duck reovirus samples, that is, confirmed isolated and purified viruses.
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