The development of a quantitative PCR ELISA to determine HCMV DNAaemia levels in heart, heart/lung and lung transplant recipients

The adaptation of the polymerase chain reaction (PCR) enzyme-linked immunos orbent assay (ELISA) using a co-amplified DNA standard to quantitate the hu man cytomegalovirus (HCMV) glycoprotein B (gB) gene in clinical samples is described. The PCR ELISA is a solution hybridisation system with colorimetr ic end stage detection of amplicons. A DNA internal standard (IS) was desig ned by replacing a probe sequence used currently within the gB region with a heterogeneous sequence, allowing co-amplification with the same oligonucl eotide primer sets and differentiation by probe hybridisation. Two DNA frag ments homologous to the gB and IS sequences were generated and used for co- amplification studies to construct a standard curve. From this the copy num ber of the gB gene present in clinical samples could be interpolated. Go-am plification with 1000 IS copies allowed quantitation of 10-1 000 000 gB DNA copies in a single PCR. This assay was validated subsequently using blood samples tested by the HCMV antigenaemia assay and showed a trend of increas ing HCMV DNAaemia with increasing antigenaemia levels. This rapid assay avo ids using gel electrophoresis and cumbersome quantitative systems. It has t he potential for early identification of patients at high risk of developin g HCMV disease, and for therapeutic monitoring. (C) 1999 Elsevier Science B .V. All rights reserved.