Primers (Invitrogen, Carlsbad, CA) and a fluorescent probe (IDT, Coralville, IA) for the HSV-1 DNA polymerase gene were used. The forward and reverse primer sequences for HSV were 5′-CATCACCGACCCGGAGAGGGAC-3′ and 5′-GGGCCAGGCGCTTGTTGGTGTA-3′, respectively. The fluorescent probe sequence was 5′-6FAM-CCGCCGAACTGAGCAG-ACACCCGCGC-BHQ-1-3′. All reactions had a final volume of 50 μL, which consisted of 10× PCR buffer (Invitrogen), 50 mM MgCl2, 2 μM of each primer, 10 mM dNTP (dATP, dCTP, dGTP, dTTP), 2 μM of the probe, 1 U/μL DNA polymerase (Platinum Taq; Invitrogen), and 10 μL DNA extract. The reactions were performed in 96-well plates (Bio-Rad, Hercules, CA), which were centrifuged for <1 minute at 1000g at room temperature in a swing-out rotor (CRU 5000 centrifuge; Damon/IEC, Needham, MA) to remove any air bubbles. Amplification and detection were performed by a PCR detection system (iCycler iQ; Bio-Rad) using the following protocol: one cycle at 95°C for 3 minutes and 45 cycles at 95°C for 30 seconds, at 55°C for 30 seconds, and at 72°C for 30 seconds. Real-time fluorescence data were collected during the annealing step. In the PCR assays, three or four negative controls were included. Three or four positive controls were included in triplicate. These controls were also run at different serial dilutions (2- or 10-fold). The PCR assay was performed in a designated room separate from the extraction room.