Primer for rapidly detecting carp edema virus and method thereof
A carp, rapid technology, applied in the direction of biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., to achieve the effect of optimizing reaction conditions and avoiding pollution
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[0030] Example 1
[0031] 1. Extraction of virus
[0032] The fish tissues infected with carp edema virus were extracted according to the instructions of the TIANamp Genomic DNAKit kit from TIANGEN.
[0033] 2. Design of primers
[0034] According to the sequence published by CEV (GenBank: MG550029.1) in GenBank, PCR primers were designed to amplify the P4a sequence. The reaction system is: 20μL rTaq enzyme, 2μL upstream primer (SEQ ID NO: 1), 2μL downstream primer (SEQ ID NO: 2), 2μL template DNA (DNA extracted in step 1), and dH 2 O 14μL. The amplification procedure is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 30 sec, a total of 35 cycles; extension at 72°C for 10 min; storage at 12°C.
[0035] The PCR target product was recovered by agarose gel electrophoresis, in accordance with OMEGA E.Z.N.A. GelExtraction Kit kit instructions for recovery.
[0036] 3. Cloning of the target gene
[0037] Mix 4.5 μL of ...
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