Multiplex-nested RT-PCR to evaluate latent and lytic Epstein Barr virus gene expression.
This paper describes the development of two multiplex-nested RT-PCR devised to evaluate latent/immortalizing (EBNA1, EBNA2, LMP1 and LMP2) and lytic [immediate early (Zebra), early, and late (VCA), respectively] Epstein Barr virus (EBV) transcripts. Subsequently, the assays have been validated evaluating the EBV latent/lytic gene expression in peripheral blood mononuclear cells (PBMC) from immunocompetent subjects (children with primary EBV infection, past EBV infection and no EBV infection) and from immunosuppressed patients (30 asymptomatic renal transplant recipients and 4 liver transplant patients with diagnosed post-transplant lymphoproliferative disorders [PTLD]). Our two multiplex-nested RT-PCR assays provide a reliable, rapid and sensitive system, enabling the simultaneous detection and identification of seven latent/immortalizing and lytic EBV transcripts. These assays could be employed in further investigations in order to evaluate the EBV transcriptional profile in EBV-related diseases both in immunocompetent and immunocompromised hosts.
Bergallo
Massimiliano
M
Department of Public Health and Microbiology, Virology Unit, University of Turin, Via Santena 9, 10126 Torino, Italy.
Costa
Cristina
C
Baro
Sara
S
Musso
Tiziana
T
Balbo
Luciano
L
Merlino
Chiara
C
Cavallo
Rossana
R
D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002648:Child; D020031:Epstein-Barr Virus Infections / Q000175:diagnosis / Q000821:virology; D005260:Female; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017735:Virus Latency / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*