Evaluation of two set of primers for detection of immediate early gene UL123 of human cytomegalovirus (HCMV).
Human cytomegalovirus (HCMV) is a widespread agent causing a life-long persistent and generally asymptomatic infection in immunocompetent individuals. In contrast, immunocompromised subjects are the most susceptible group to experience HCMV disease. First genes to be expressed during the replication cycle are the immediate early (IE) genes, the products of which have pleiotropic effects on host cell metabolism. Aim of this study was to compare two set of primers in the optimization and standardization of a RT-PCR assay for qualitative detection of mRNA encoded by the IE gene UL123 (IE1). The RT-PCR assays were then used to evaluate the UL123 gene expression in 29 peripheral blood leukocyte (PBL) samples obtained from 14 renal transplant recipients. In particular, 21/29 (72.4%) were positive with set A of primers vs. 24/29 (82.8%) with set B. Only one sample were negative with set B and positive with set A. Twenty-four of 29 samples (82.8%) were pp65-antigaenemia positive: 21 mRNA-UL123 positive with set A vs. 22 with set B; all viraemia-positive patients were mRNA-UL123 positive with both set A and B. Five of 29 samples were pp65-antigaenemia negative: 1 mRNA-UL 123 positive with set A vs. 2 with set B; all of them were viraemia-negative. These two RT-PCR assays could provide a reliable, rapid and sensitive system enabling the detection and identification of UL123 transcripts and could be usefully employed to study the pathogenesis of HCMV-related diseases.
Bergallo
Massimiliano
M
Department of Public Health and Microbiology, Virology Unit, University of Turin, Via Santena 9, Torino 10126, Italy.
Costa
Cristina
C
Terlizzi
Maria Elena
ME
Margio
Samuela
S
Sidoti
Francesca
F
Sinesi
Franca
F
Cavallo
Rossana
R
D000328:Adult; D001483:Base Sequence; D001709:Biotechnology; D003587:Cytomegalovirus / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000209:etiology / Q000821:virology; D017931:DNA Primers / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D017781:Genes, Immediate-Early; D005814:Genes, Viral; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects; D008297:Male; D008875:Middle Aged; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral