DETECTION OF HERPES-SIMPLEX VIRUS TYPE-1 BY AN IN-SITU POLYMERASE CHAIN-REACTION TECHNIQUE

The purpose of our study was to develop a method for detecting herpes simplex virus (HSV) DNA that combined the high sensitivity of the poly merase chain reaction (PCR) with the precise anatomical localization p rovided by in situ hybridization (ISH). We used insitu PCR (ISPCR), IS H, and standard PCR methods to determine the proportion of Vero cells carrying HSV-1-specific DNA before and after 1, 2, and 4 h of infectio n with HSV-1 or with HSV-2. Uninfected Vero cells and Vero cells infec ted with HSV-2 were never found to be positive for HSV-1 DNA by either ISPCR, ISH, or PCR. In contrast, using ISPCR, HSV-1 infected Vero cel ls showed an increase in the percentage of cells containing HSV-1 DNA from 20% at 1 h to 76% at 4 h after infection. Comparing the ISPCR res ults with ISH and standard PCR demonstrated that ISPCR was markedly mo re sensitive than ISH; in fact, the sensitivity of in situ PCR was sim ilar to that seen with standard PCR. These results demonstrate that IS PCR is a highly sensitive method for amplifying genomic DNA sequences within intact single cells. This technique combines the exquisite sens itivity of conventional PCR technology with the precise cellular local ization afforded by ISH. In addition, it allows for an accurate quanti tative determination of the number of virally infected cells.