Quantification of human cytomegalovirus DNA using the polymerase chain reaction

Abstract

The important goal of developing quantitative assays for viral nucleic acids in clinical samples has been achieved for human cytomegalovirus (HCMV) by using a modified polymerase chain reaction (PCR). A control PCR target sequence was constructed by PCR mutagenesis to allow the post-amplification quantification of HCMV DNA. The control region was identical to a naturally occurring sequence within the glycoprotein B (gB) coding part of the virus genome, except that a unique restriction site, introduced by the aforementioned mutagenesis step, allowed post-amplification differentiation of control/non-control target amplified product. This technique was initially validated using known amounts of cloned control/non-control target DNA, and was found to be sufficiently sensitive to allow the quantification of a range of 10 to 10(6) genome equivalents of virus. The method was applied to urine samples of congenitally infected infants for which infectious virus titres were available. The results obtained demonstrated that the number of infectious virions determined by conventional cell culture represented a small proportion of the HCMV genome present in the samples, as assessed by the quantitative PCR methodology.