Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of Human Cytomegalovirus in clinical specimens

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.