title: A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.
abstract: Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.
authors: Unit of Human Virology and Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
authors: Francesca
authors: F
authors: Gatto
authors: Giulia
authors: G
authors: Cassina
authors: Francesco
authors: F
authors: Broccolo
authors: Giuseppe
authors: G
authors: Morreale
authors: Edoardo
authors: E
authors: Lanino
authors: Eddi
authors: E
authors: Di Marco
authors: Efthiya
authors: E
authors: Vardas
authors: Daniela
authors: D
authors: Bernasconi
authors: Stefano
authors: S
authors: Buttò
authors: Nicola
authors: N
authors: Principi
authors: Susanna
authors: S
authors: Esposito
authors: Gabriella
authors: G
authors: Scarlatti
authors: Paolo
authors: P
authors: Lusso
authors: Mauro S
authors: MS
authors: Malnati
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis*; D005260:Female; D004854:Herpesvirus 4, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D060885:Multiplex Polymerase Chain Reaction; D010949:Plasma / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*; D014766:Viremia / Q000175:diagnosis
chemical_list: D004279:DNA, Viral