Quantitative Detection of HSV-1 DNA

The viral DNA was extracted using phenol/chloroform solution and precipitated from the organic phase. The procedures were published previously in [28 (link)]. The DNA pellet was washed twice in a solution containing 0.1 M trisodium citrate in 10% ethanol, and then, dissolved in 8 mM NaOH. The concentration of DNA was determined via fluorometer analysis using a Qubit double-stranded DNA (dsDNA) HS (High Sensitivity) Assay Kit according to the manufacturer’s instructions. The amplification of viral DNA 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 reverse (10 µM) primers (Fw 5′-catcaccgacccggagagggac; Rev 5′-gggccaggcgcttgttggtgta), and a TaqMan probe (5 µM) (5′-6FAM-ccgccgaactgagcagacacccgcgc-TAMRA, where 6FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine). The amplification was carried out using the Applied Biosystems 7300 Real-Time PCR System (Foster City, CA) 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 the concentration in µg of DNA/µL.