1.  Qiong Li a,b,d, Zhiqin Yuea, ∗, Hong Liuc, Chengzhu Lianga, Xiaolong Zhenga, Yuran Zhaoa, Xiao Chena, Xizhi Xiaoa, Changfu Chenb Journal of Virological Methods 163 (2010) Marine Virology LAB Myunglip Lee

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3. Virus?

5. Lymphocystis Disease ? Lymphocystis is a common, chronic and benign infection caused by an iridovirus that results in uniquely hypertrophied cells, typically in the skin and fins of only the more advanced orders of fishes. Signs and pathologic changes ? Lymphocystis is a terminal infection of individual cells, and the effect on the host is generally slight. The virus is typically dermatotropic and superficial; the condition is much like that of warts, in that the lesions are macroscopic and occur mostly at the periphery of the vascular system.

6. External signs Cauliflower-like lesions on body surface, including mouth region, fin and tail region

8. Materials and methods 50 fish 53 fish 6 fish

9. Materials and methods DNA extraction Brain Kindney Spleen homogenized Takara universal g enomic DNA extra ction kit

10. Materials and methods Design of primers for the LCDV LAMP assay Fig. 1. Primer design for the detection of LCDV using the LAMP assay. (A) Partial DNA sequence of the MCP fragment of LCDV (accession no. AY380826) showing the target region for the LAMP assay. The inner and outer LAMP primers designed from this region are indicated by different background. (B) A schematic diagram showing the positions at which the primers attach for target gene amplification.

11. Materials and methods  Analysis of the LAMP products 2% Agarose gelSYBR Green IObserve  Specificity of the LCDV LAMP assay LCDV DNA was evaluated by testing EHNV, TFV, BIV, STIV, ISKNV, RSIV,WSSV

12. Materials and methods Sensitivity of the LCDV LAMP assay DNA Paralichthys olivaceus 10­¹ to 10­⁸ (DNA) Ten-fold serial dilutions LAMP PCR real-time quantitative PCR The conventional PCR reaction was carried out in a 25 μ L reaction mixture containing 12.5 μ L 2× Premix Taq (Takara), 0.4Meach of F3 and B3, 2.0 μ L of DNA samples, and 8.5 μ L of distilled water. The reaction mixture was incubated at 94 ◦C for 5min, followed by 40 cycles of 94 ◦C for 30 s, 58 ◦C for 1min, 72 ◦C for 1min, and a final cycle at 72 ◦C for 10 min. The reaction mixture contained 2 μ L DNA template, 12.5 μ L of Taqman universal PCR master mixture (AppliedBiosystems), 0.2Mof each primer (forward primer: 5CAAGCTATACAATCCAATTACACCAGTT-3; reverse primer: 5-CAGCAGCAATACCCGGTAAATC-3), and 1L of 25× SYBR Green I in a total volume of 25 μ L. Amplifications were performed with the following profile: 2min at 50 ◦C, denaturation at 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 s and 60 ◦C for 1min.

14. Results Optimization of the LCDV LAMP reaction Fig. 2. Determination of the optimal temperature for the LAMP assay by agarose gel electrophoresis (A) and SYBR Green I staining (B). M: Molecular marker; 1–7: LAMP reactions at 59, 60, 61, 62, 63, 64, and 65 ◦C, respectively; 8: blank control.

15. Results Optimization of the LCDV LAMP reaction (A) Agarose gel showing the effect of varying the amplification time in the LCDV LAMP assay using 4 primers (FIP, BIP, F3, and B3) at 63 ◦C for 25–95 min. M: Molecular marker; 1–9: reactions at 25, 35, 45, 55, 60, 65, 75, 85, and 95 min, respectively; lane 10: blank control. The LAMP assay detected LCDV after 75 min. (B) Agarose gel showing the effect of varying the amplification time in the LCDV LAMP assay using 5 primers (FIP, BIP, F3, B3, and loop-B) at 63 ◦C for 25–95 min. M: Molecular marker; 1–9: reactions at 25, 35, 45, 55, 60, 65, 75, 85, and 95 min, respectively; 10: blank control. The LAMP assay detected LCDV as early as 35 min. 65min 35min

16. Results Specificity of the LCDV LAMP assay Fig. 4. Determination of the specificity of the LAMP assay by agarose gel electrophoresis (A) and SYBR Green I staining (B). M: Molecular marker; 1 and 2: LCDV, 3–9: epizootic hematopoietic necrosis virus (EHNV), tiger frog virus (TFV), Bohle iridovirus (BIV), soft-shelled turtle iridovirus (STIV), infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), and WSSV genomic DNA; 10: blank control.

17. Results Fig. 5. Determination of the sensitivity of LAMP (A), PCR (B), and real- time quantitative PCR(C) using 10-fold serial dilutions of LCDV DNA as templates; M: DL2000 DNA marker; 1: non-diluted LCDV DNA; 2–9: 10−1 to 10−8 dilutions of LCDV DNA; 10: blank control. Sensitivity of the LCDV LAMP assay

18. Applicability of the LCDV LAMP assay Results Table 1. Detection of LCDV by LAMP and conventional PCR from infected, unknown and healthy samples.

20. S.iniae and LCDV infections pose a serious threat to the aquaculture industryand are responsible for significant financial losses. It is important to establish simple, rapid, and reliable methods for the diagnosis of S.iniae, LCDV to prevent further disease transmission or outbreaks. Discusson This is the first report detailing the use of the LAMP technique for molecular diagnosis of S. iniae and LCDV.