Journal of General Virology (1992), 73, 2405-2408. Printed in Great Britain 2405 Quantification of human cytomegalovirus DNA using the polymerase chain reaction Jayne C. Fox, Paul D. Griffiths and Vincent C. Emery* University Division of Communicable Diseases, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K. 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 quantifica- tion of HCMV DNA. The control region was identical to a naturally occurring sequence within the glycopro- tein B (gB) coding part of the virus genome, except that a unique restriction site, introduced by the aforemen- tioned 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 l0 s 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 repre- sented a small proportion of the HCMV genome present in the samples, as assessed by the quantitative PCR methodology. Since the initial description of the polymerase chain reaction (PCR) by Saiki and co-workers in 1988, the PCR has been increasingly applied to the detection of a variety of infectious agents within clinical material. The PCR can be utilized to detect both DNA and, following a reverse transcription step, RNA target sequences. Although replica PCRs on endpoint diluted samples (Simmonds et al., 1990) does allow the quantitative detection of viral sequences, the majority of PCR applications are qualitative, i.e. the presence of the target sequence in the sample of interest will yield an amplified DNA fragment and hence a positive result. However, in many circumstances there are diagnostic and prognostic implications of being able to quantify the amount of virus present in a particular clinical specimen. For example, the prognosis of neonates congenitally infected with human cytomegalovirus (HCMV) is directly related to the amount of HCMV present in their urine at the time of presentation (Stagno et al., 1986). Likewise, in patients infected with human immunodefi- ciency virus (HIV), progression to AIDS is accompanied by an increase in plasma virus load (Clark et al., 1991). In such situations, conventional cell culture and TCIDso determinations are of great benefit and cannot be replaced by existing qualitative PCR technology. In the PCR system, amplification of target DNA initially occurs in an exponential fashion but ultimately the efficiency of amplification decreases owing to a number of factors, including limiting reagents and inefficiency of primer binding in the later cycles. Several methodologies have been investigated in an attempt to perform quantitative PCR, including the use of a co- amplified sequence, e.g. fl-globin (Pang et al., 1990), that is amplified simultaneously with the target sequence. Solution hybridization following PCR has also been investigated (Kellog et al., 1990), as has the use of limiting dilution of the sample followed by PCR (Simmonds et al., 1990). The latter method is accurate but time-consuming, expensive to perform and is unlikely to be suitable for the analysis of many samples, as would be required in a diagnostic laboratory or in studies involving large numbers of clinical specimens. The methods which use control primers that amplify other gene products or which use known amounts of target DNA in concurrent PCRs offer only a semi- quantitative PCR methodology since they cannot com- pensate for local variation in PCRs or the possible presence of inhibitory substances within specific sam- pies. In addition, such methods do not compensate for the efficiency of the reverse transcriptase step in the amplification of RNA target sequences. Ideally, a quantitative PCR should compensate for all these variable (i.e. primer binding efficiency, inhibitory substances in the samples and variations between sample 0001-0697 © 1992SGM