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Figure and table legends
Figure 1. Detection of Megalocytivirus Korean isolates using the diagnostic method according to the OIE manual. Lane M: marker, Lane 1–16, 17–21: Korean RSIV isolates, Lane 22: positive control, Lane N: negative control.
Figure 2. Comparison of the primer region for Megalocytivirusdetection using the CLC Main Workbench software (Ver. 8.1.0). Sequence variation in OIE primers (A, B). A red-dot box indicates the variation within the primer region.
Figure 3. Comparison of the primer region for Megalocytivirusdetection using the CLC Main Workbench software (Ver. 8.1.0). Sequence variation in Meg-Univ (A), TRBIV-specific (B), and RSIV-specific (C) primers. A red-dot box indicates the variation within the primer region. TRBIV, turbot reddish body iridovirus; RSIV, red seabream iridovirus.
Figure 4. Detection of two genotypes of Megalocytivirus according to the newly designed multiplex PCR primers using a plasmid construct. Lane 1: Meg-Univ, Lane 2: TBRIV-specific, Lane 3: RSIV-specific, Lane 4: Meg-Univ and TBRIV-specific, Lane 5: Meg-Univ and RSIV-specific, Lane 6: Meg-Univ, TBRIV specific, and RSIV specific. TRBIV, turbot reddish body iridovirus.
Figure 5. Detection of serially diluted samples ofMegalocytivirus primers plasmid construct. Lane M: 100-bp marker, Lane 1–9: 2.0 × 109–2.0 × 101 copy number plasmid, Lane N: negative control. (A) Serial dilution of Meg-Univ -specific, (B) serial dilution of TBRIV-specific, (C) serial dilution of RSIV-specific, (D) serial dilution of Meg-Univ and TBRIV-specific, (E) serial dilution of Meg-Univ and RSIV-specific. TRBIV, turbot reddish body iridovirus.
Figure 6. Specificity of diverse Megalocytivirus primers. (A) Specificity test of RSIV multiplex PCR primers using different virus strains. Lane M: marker, Lane 1–6: virus strains; 1: SVC; 2: VER; 3: VHS; 4: 4: IHN; 5: LCDV; 6: positive control (Meg-Univ+TRBIV-specific); 7: positive control (Meg-Univ+RSIV-specific), Lane N: negative control. (B) Specificity test of virus strains using specific primers of virus strains. Lane M: marker, Lane 16: virus strains; 1, 3, 5, 7, 9: LCDV, SVC, VER, VHS, IHN, respectively; Lane 2, 4, 6, 8, 10: Negative control for each virus strain. SVC, spring viraemia of carp; IHN, infectious hematopoietic necrosis; VHS, viral hemorrhagic septicemia; VER, viral encephalopathy and retinopathy; LCDV, lymphocystis virus; TRBIV, turbot reddish body iridovirus.
Figure 7. Optimization of the annealing temperature for multiplex PCR assay. (A) Meg-Univ (534 bp) and TBRIV-specific (451 bp), (B) Meg-Univ (534 bp) and RSIV-specific (245 bp). Lane M: marker, (1): 50ºC; (2): 52.0ºC; (3): 54.5ºC; (4): 55.8ºC; (5): 58.4ºC; (6): 60.3ºC; (7): 62.2ºC; (8): 64.0ºC; 9: 65.9ºC; 10: 67.8ºC; 11: 70.2ºC. TRBIV, turbot reddish body iridovirus; RSIV, red seabream iridovirus.
Figure 8. Optimization of the primer concentration for multiplex PCR assay. (A) Meg-Univ (534 bp) and TBRIV-specific (451 bp), (B) Meg-Univ (534 bp) and RSIV-specific (245 bp). Lanes 1–11 indicate the concentration combination of three primers specific for Meg-Univ, TBRIV-specific, and RSIV-specific. (1) 30:10:10 pmol/μL, (2) 20:10:10 pmol/μL, (3) 10:10:10 pmol/μL, (4) 30:20:10 pmol/μL, (5) 30:30:10 pmol/μL, (6) 30:10:20 pmol/μL, (7) 30:10:30 pmol/μL, (8) 15:10:10 pmol/μL, (9) 15:5:5 pmol/μL. TRBIV, turbot reddish body iridovirus; RSIV, red seabream iridovirus.
Figure 9. Detection of red seabream iridovirus (RSIV) Korean isolates using the newly designed multiplex PCR primers. Lane M: marker, Lane 1–16, 17–21: Korean RSIV isolates, Lane 22: positive control (Meg-Univ+TRBIV-specific), Lane 23: Meg-Univ+RSIV-specific), Lane N: negative control. TRBIV, turbot reddish body iridovirus.
Table 1. Megalocytivirus isolated from fish samples in Korea from 2012 to 2018 TRBIV, turbot reddish body iridovirus; RSIV, red seabream iridovirus.
Table 2. Primers used for nucleotide analysis of Megalocytivirusgenes RSIV, red seabream iridovirus.
Table 3. Primers used for Megalocytivirus detection RSIV, red seabream iridovirus; TRBIV, turbot reddish body iridovirus
Figures
Figure 1