title: Multiplex real-time PCR assay for simultaneous quantitation of human cytomegalovirus and herpesvirus-6 in polymorphonuclear and mononuclear cells of transplant recipients.
abstract: Human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) are two closely related viruses, which belong to the Herpesviridae family. Following primary infection, they are thought to persist for life as latent forms in mononuclear cells. HCMV and HHV-6 can cause considerable morbidity in immunocompromised individuals, such as transplant patients. A sensitive and specific LightCycler multiplex real-time PCR assay based on fluorescence energy transfer (known as FRET) was developed. This assay, by using two sets of hybridization probes specific for HHV-6 (A and B) and HCMV, can differentiate reliably and quantify simultaneously both viruses in order to diagnose reactivation processes. The assay was optimized and the lower limit of detection for both viruses was determined to be 10 viral genome copies per reaction. Both viruses were quantified in 83 peripheral blood mononuclear cells (PBMCs) and 87 polymorphonuclear leukocytes (PMNLs) collected from 32 transplant recipients. This multiplex real-time quantitative PCR was finally compared with two other quantitation and detection assays used daily in laboratory (PCR DIG detection and antigenemia for HCMV, TaqMan Assay for HHV-6). This technique can be useful for the differentiation and quantitation of HCMV and HHV-6 for monitoring transplant patients.
authors.affiliation: Laboratoire de Virologie, CHU Dupuytren, Limoges, France.
authors.forename: Karine
authors.initials: K
authors.lastname: Pradeau
authors.forename: Ludovic
authors.initials: L
authors.lastname: Couty
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Szelag
authors.forename: Pascal
authors.initials: P
authors.lastname: Turlure
authors.forename: Florence
authors.initials: F
authors.lastname: Rolle
authors.forename: Pierre
authors.initials: P
authors.lastname: Ferrat
authors.forename: Dominique
authors.initials: D
authors.lastname: Bordessoule
authors.forename: Yann
authors.initials: Y
authors.lastname: Le Meur
authors.forename: François
authors.initials: F
authors.lastname: Denis
authors.forename: Sylvie
authors.initials: S
authors.lastname: Ranger-Rogez
mesh_terms: D000328:Adult; D000956:Antigens, Viral / Q000097:blood; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis / Q000097:blood / Q000235:genetics; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000821:virology*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014180:Transplantation
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D015345:Oligonucleotide Probes