Fast buffer 1

PCR amplification was performed in a total volume of 20 μL containing 2 μL of 10 × Fast Buffer I (Takara, China), 1 μL of dNTP mixture (100 μM each, Takara), 0.2 μL of SpeedStar HS Taq DNA polymerase (5 U/μL, Takara), 2 μL of Fl-dUTP (25 nM, Perkin Elmer), 0.2 μL of identification primer (10 μM, Sangon), 0.5 μL of 10 mg/mL bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA), 1 μL of 20% Polyvinyl pyrrolidone 40 (Sigma-Aldrich, St. Louis, MO, USA) and a total of 10 ng of DNA. Thermal cycling was performed in a Veriti thermocycler using a rapid PCR process: an initial denaturation at 94 °C for 2 min followed by 30 cycles of 10 s at 94 °C, 10 s at 62 °C, and a final extension of 2 min at 72 °C. After the reaction cycle, the reaction products were held at 4 °C.

Quantitative real-time PCR was performed using a microfluidic real-time PCR system (GeneSoC®, Kyorin, Tokyo, Japan). Gene SoC® can show three colors; therefore, simultaneous measurements of HSV DNA (Cy5) and VZV DNA (FAM) were performed in this study. Since the time required for real-time PCR was approximately 12 min, the HSV and VZV DNA test results were available in 40 min following the collection of tears.
The PCR mix contained cold lysis buffer including DNA extracts 200 μL, HSV-1 primer & probe [Probe (Cy5): TAG TGG GCC TCC ATG GG, HSV1F: GGG CCG TGA TTT TGT TTG TC, HSV1R: CCG CCA AGG CAT ATT TGC] 2.5 μL, HHV3 TaqMan probe (FAM) (TaqMan gene expression assay, Life Technologies Japan, Carlsbad, Tokyo, Japan) 1.0 μL, 10 × FAST Buffer I (Takara Bio Inc, Shiga, Japan) 2.0 μL, dNTP Mixture (Takara Bio) 1.6 μL, SpeedSTAR™ HS DNA Polymerase (Takara Bio) 0.4 μL, and distilled water 3.9 μL.