· 2.9. Challenge. At the time point of 21 days post-vaccination, fish of vaccinated and control groups (each group, n = 30) were transferred to new tanks and challenged by injected intramuscularly with 4.0 × 10 4 TCID 50 of live GCRV in saline buffer, and then mortality rates were recorded daily in 20 days after viral challenge. All experiments were performed for three replicates.
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· * Water Contamination
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· Each reaction tube contained 10 μL 2 × SGExcel FastSYBR Mixture (With ROX, Sangong Biotech, Shanghai, China), 8.6 μL dH 2 O, 0.2 μL of each forward and reverse primer (1 pmol/μL), and 1 μL of DNA template. Molecular grade water was used as a negative control. Triplicate PCR reactions were carried out for all samples and negative controls.
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The water-soluble gas content (in the standard state) is also calculated by a numerical simulation method, and the formula is as follows (8) where V s is the water-soluble gas content (m 3 /t), S is the solubility (m 3 methane/m 3 water), M e is the moisture content (%), and ρ w is the density of water ….
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