A novel, internally competitive polymerase chain reaction for quantification of Human Cytomegalovirus DNA in human leukocytes

Abstract

The rapid, low cost estimation of Human Cytomegalovirus (HCMV) viral load in the blood of immunosupressed individuals is crucial for the timely diagnosis of transplant recipients with clinically significant HCMV infections requiring treatment. To this end we have developed a novel polymerase chain reaction (PCR) assay with internal controls for competitive quantification of cytomegalovirus DNA in human leukocytes. The reaction simultaneously amplifies, under reduced stringency conditions, a 136 bp fragment of the HCMV LA gene together with a single anonymous 200 bp fragment of human DNA using a single-primer pair. The primers used are specific for the HCMV gene sequence but contain a single mismatch that permits the amplification of the competing human DNA fragment. Quantification is achieved by comparison of the amount of the two products. The assay quantifies between 103and 106HCMV genomes in 106leukocytes, a range that permits low-level clinically unimportant HCMV infections to be distinguished from those likely to cause serious disease. The advantages of the method are that no external controls are needed, quantification is achieved from a single reaction and no separate measurement of host DNA concentration is required.