Quantitative Detection of HSV-1 DNA

The viral DNA was extracted with phenol/chloroform solution and precipitated from the organic phase. The procedures were published previously [32 (link)]. The DNA pellet was washed twice in a solution containing 0.1 M trisodium citrate in 10% ethanol and dissolved in 8 mM NaOH. The DNA concentration was determined by fluorometer analysis with the Qubit double-stranded DNA (dsDNA) HS (High Sensitivity) Assay Kit according to the manufacturer’s instructions. Viral DNA amplification was carried out using TaqMan™ Universal Master Mix II (Applied Biosystems™, Foster City, CA, USA) in a 50 µL reaction mixture containing TaqMan Universal Master Mix II, DNA (100 ng), HSV-1 forward (10 µM), and HSV-1 reverse (10 µM) primers, TaqMan probe (5 µM). The sequence of primers is shown in Table 2. The amplification was carried out on Applied Biosystems 7300 Real-Time PCR System under the following conditions: 10 min at 95 °C, 60 s at 95 °C for 40 cycles, 30 s at 60 °C, and 30 s at 72 °C. Absolute quantification Real-time PCR using a specific TaqMan probe was performed to detect viral DNA. Viral load was derived from the threshold cycle (CT) using the standard curve generated in parallel, and the result is expressed as a concentration in µg of DNA/µL.