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

The rapid, low cost estimation of Human Cytomegalovirus (HCMV) viral load i n the blood of immunosupressed individuals is crucial for the timely diagno sis of transplant recipients with clinically significant HCMV infections re quiring treatment. To this end we have developed a novel polymerase chain r eaction (PCR) assay with internal controls for competitive quantification o f cytomegalovirus DNA in human leukocytes. The reaction simultaneously ampl ifies, under reduced stringency conditions, a 136 bp fragment of the HCMV L A 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 seque nce but contain a single mismatch that perm its the amplification of the co mpeting human DNA fragment. Quantification is achieved by comparison of the amount of the two products. The assay quantifies between 10(3) and 10(6) H CMV genomes in 10(6) leukocytes, 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 separ ate measurement of host DNA concentration is required. (C) 1999 Academic Pr ess.