Multiplex polymerase chain reaction for the evaluation of cytomegalovirus DNA load in organ transplant recipients

Because of the considerable impact of human cytomegalovirus (HCMV) infectio n, sensitive, specific, and standardized methods are required for rapid and accurate evaluation of viral load in monitoring transplant recipients. The aim of the present study was to evaluate the usefulness of a multiplex pol ymerase chain reaction (PCR) for the coamplification of HCMV-DNA and beta-g lobin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassa y (DEIA), which is based on the hybridization of amplified DNA with a singl e-stranded DNA probe, which coats microtitre wells. Colorimetric detection of the DNA-antibody complex was carried out and optical density (O.D.) was recorded at 450/630 nm. To quantify HCMV/DNA load, a standard curve to whic h samples O.D, refer was obtained by amplifying serial dilutions of recombi nant PGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 3 40 PMNL specimens from 102 solid organ recipients were tested for the detec tion of pp65 antigen and HCMV-DNA. The results showed a flood correlation b etween viral load and clinical symptoms of HCMV infection; high specificity and predictive values for HCMV disease were found by PCR, using a cut-off limit of 10(3) genomic copies per 2 x 10(5) PMNL. These findings indicate t hat the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transpla nted patients. (C) 2000 Wiley-Liss, Inc.