The purpose of our study was to develop a method for detecting herpes simplex virus (HSV) DNA that combined the high sensitivity of the polymerase chain reaction (PCR) with the precise anatomical localization provided by in situ hybridization (ISH). We used in situ PCR (ISPCR), ISH, 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 infection with HSV-1 or with HSV-2. Uninfected Vero cells and Vero cells infected 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 cells 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 results with ISH and standard PCR demonstrated that ISPCR was markedly more sensitive than ISH; in fact, the sensitivity of in situ PCR was similar to that seen with standard PCR. These results demonstrate that ISPCR is a highly sensitive method for amplifying genomic DNA sequences within intact single cells. This technique combines the exquisite sensitivity of conventional PCR technology with the precise cellular localization afforded by ISH. In addition, it allows for an accurate quantitative determination of the number of virally infected cells.
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