Detection of mRNA from the immediate early gene of human cytomegalovirus in infected cells by in vitro amplification

Abstract

It would be desirable to have a facile means of detecting the presence of viral DNA or mRNA in any given biological sample, especially in view of the growing immunocompromised population in which the diagnosis of viral disease is a common problem. In vitro amplification of DNA using methods such as the polymerase chain reaction, offers a sensitive means of detecting both DNA and mRNA. We have used the polymerase chain reaction to detect DNA and mRNA from human cytomegalovirus infected human fibroblasts. Viral mRNA was differentiated from DNA using primers which flank a splice junction, resulting in a smaller product for the mRNA template. A cDNA was prepared from total RNA using a primer specific for the gene of interest, in this case the major immediate early transcript of human cytomegalovirus. The cDNA was then amplified using a modified polymerase chain reaction protocol. mRNA from the major immediate early gene was detected in cultured fibroblasts as early as 6 h after infection, and continued to be expressed for at least 96 h post infection. Sensitive and facile detection of viral mRNA should facilitate diagnostic and basic studies of viral pathogenesis.