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

Abstract

Because of the considerable impact of human cytomegalovirus (HCMV) infection, 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 polymerase chain reaction (PCR) for the coamplification of HCMV-DNA and β-globin genomic sequence in polymorphonuclear leukocytes (PMNL). Analysis and quantification of PCR products were carried out by a DNA enzyme immunoassay (DEIA), which is based on the hybridization of amplified DNA with a single-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 which samples O.D. refer was obtained by amplifying serial dilutions of recombinant PGEM-3Z plasmid DNA containing a genomic fragment of glycoprotein B. 340 PMNL specimens from 102 solid organ recipients were tested for the detection of pp65 antigen and HCMV-DNA. The results showed a good correlation between vital 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 103 genomic copies per 2 x 105 PMNL. These findings indicate that the system described is an efficient and reproducible diagnostic method easy to apply for routine diagnosis and therapeutic monitoring of transplanted patients. (C) 2000 Wiley-Liss, Inc.