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Figure 1 .Virus RNA was purified from 10E1-10E4 copy number of Red Spotted Grouper Nervous Necrosis Virus (RGNNV) using the Geneaid Virus DNA/RNA Kit II (3 replications of each copy number). The purified RNA was eluted with 30 μl RNase-free Water. cDNA synthesis was carried out with a 10 μl aliquot of purified RNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche) in a final volume of 20 μl. A Real-time PCR assay was then performed with 3 μl of synthesized cDNA as template, primers (designed to amplify the T4 region on the RNA2 segment), and Fast SYBR Green PCR Master Mix using the StepOnePlusTM Real-Time PCR system (Applied Biosystems). The results confirmed that virus RNA can be successfully extracted and detected from as low as 10E1 copy number of RGNNV. The average cycle threshold (Ct): 10E4 = 23.88, 10E3 = 27.72, 10E2 = 31.22, 10E1 = 34.62. The low Ct values indicate a high number of target nucleic acid in the sample.

Figure 3 .HBV (DNA), HCV (RNA), HIV (RNA), and HTLV (RNA) were purified from 200 µl of positive clinical serum samples using the Viral Nucleic Acid Extraction Kit II. Real-time qPCR and 1-step qRT-PCR reactions were then conducted using the ABI 7300 Sequence Detection System (3 replications of each copy number). Serum samples containing various amounts of DNA/RNA viruses ranging from 10E1 to 10E6 copies/ml were successfully detected and identified. The low Ct values indicate a high number of target nucleic acid in the sample.