Quantifying Virus RNA from Serum Samples

To extract RNA, a serum sample (50 μL) was added to 200 μL of diethylpyrocarbonate-treated water (Ambion, Carlsbad, CA, USA), which was then added to 750 μL of TRIzol LS Reagent (Ambion). Samples were incubated for 20 min at ambient temperature. After incubation, 200 μL of chloroform (Sigma Aldrich) was added, mixed thoroughly, and incubated for 10 min at ambient temperature. After incubation, samples were centrifuged at 12,000 × g for 15 min at 4°C. A total of 400 μL of the aqueous phase was collected, and the RNA was precipitated by adding 1 μL of GlycoBlue (15 μg/μL) (Ambion) and 400 μL of isopropanol (Sigma Aldrich). Samples were incubated at ambient temperature for 10 min and centrifuged at 12,000 × g for 10 min at 4°C. The resulting pellet was then washed in 1 mL of 75% ethanol (Sigma Aldrich) and centrifuged at 12,000 × g for 5 min at 4°C, after which the pellet was air-dried for 10 min at ambient temperature and resuspended in 50 μL of diethylpyrocarbonate-treated water. Virus RNA was quantified by using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) and primers and a probe specific for the envelope gene (bases 1188–1316) (35 (link)). A standard curve was generated against a synthetic oligonucleotide, and genome copies were expressed as copies per milliliter. The lower limit of detection was 3.0 log₁₀ copies/mL.