Detecting Herpes Viruses in Ocular Fluids

Genomic DNA of HHV in the aqueous humour and vitreous fluids was measured through the use of two independent PCR assays: (1) a qualitative multiplex PCR and (2) a quantitative real-time PCR. The result analysis for the PCR is shown in fig 1.
DNA was extracted from samples using an E21 virus minikit (Qiagen, Valencia, CA) installed on a Robotic workstation for automated purification of nucleic acids (BioRobot E21, Qiagen). The multiplex PCR was designed to qualitatively measure genomic DNA of eight human herpes viruses, that is, herpes simplex virus type 1 (HSV-1), type 2 (HSV-2), Varicella-zoster virus (VZV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), human herpes virus type 6 (HHV6), type 7 (HHV7) and type 8 (HHV8). The PCR was performed using a LightCycler (Roche, Switzerland). Primers and probes of HHV1–8 and the PCR conditions have been described previously.8 (link) Specific primers for the virus were used with Accuprime Taq (Invitrogen, Carlsbad, CA). Products were subjected to 40 cycles of PCR amplification. Hybridisation probes were then mixed with the PCR products. Subsequently, real-time PCR was performed only for the human herpes virus, with the genomic DNA detected by multiplex PCR (fig 1). The real-time PCR was performed using Amplitaq Gold and the Real-Time PCR 7300 system (ABI, Foster City, CA). The sequence of the HHV1–8 primers and probes are shown in table 1. The primers of the viruses and the PCR conditions have been described in previous reports.9 (link)^–13 (link) Our research group has also previously reported the primers of the sequences for VZV.14 (link) All of the products obtained were subjected to 45 cycles of PCR amplification. The value of viral copy number in the sample was considered to be significant, when more than 50 copies/tube (5×103 (link)/ml) were observed.