Analysis of HSV-DNA and RNA Using the Polymerase Chain Reaction

Abstract

The polymerase chain reaction (PCR) technique is a sensitive method for detection of nucleic acids that can be used to detect herpes simplex virus (HSV)-DNA and RNA in tissue samples with greater sensitivity than hybridization with specific probes (1,2). In its most basic form, PCR involves multiple cycles of denaturation of DNA, annealing with specific primers and replication of specific DNA using a thermostable DNA polymerase like Taq polymerase, resulting in amplification of a specific DNA sequence. Reverse transcriptase polymerase chain reaction (RT-PCR) employs a preliminary reverse transcription step of RNA, using either a specific 3′ or an oligo (dT) primer, to produce complementary DNA (cDNA), followed by PCR using primers specific for the transcript of interest. In its standard application, PCR offers qualitative information regarding the presence or absence of target sequences, and has been used to analyze latently infected ganglia and brain for HSV-DNA (6,8–11) and RNA (6–10,12). As described in the following, with the inclusion of mutated templates as internal standards, PCR can be used to determine a quantitative estimate of the number of HSV genomes and transcripts in tissue extracts. Histologically, in situ hybridization (ISH) can be used to detect HSV-DNA and RNA in specific cells in the nervous system (3–5), although it has not been successfully applied to detect HSV genomes during latency (6,7). PCR methods can be applied to tissue sections (in situ PCR), making it possible to identify individual cells harboring HSV genomes, even during latency (9,13).