Double-step PCR assay to quantify Epstein-Barr viral load in peripheral blood.
Posttransplant lymphoproliferative disorders (PTLD) are a severe complication arising in solid organ transplant patients. A strong correlation between Epstein-Barr virus (EBV) infection, the grade and type of immunosuppression, and the development of PTLD has been recognized. This article describes the development of a double-step polymerase chain reaction (PCR) assay for the quantification of EBV-deoxyribonucleic acid (DNA) to monitor EBV infection. Screening of samples containing >/=10(3) viral genomes/10(5) peripheral blood mononuclear cells (PBMC) or 100 micro L serum by a semiquantitative PCR assay is followed by quantification of the samples containing a high number of viral genomes in a quantitative-competitive (QC)-PCR assay. Screening by semiquantitative PCR selects samples with a high number of viral genomes for use in the more labor-intensive and expensive QC-PCR assay and thus provides a handy means for quantitative DNA analysis of large numbers of samples. Our double-step PCR assay can be employed in EBV viral load measurement in PBMC and serum samples to monitor transplanted patients at risk to develop PTLD.
Bergallo
Massimiliano
M
Department of Public Health and Microbiology, Virology Unit, University of Turin, Via Santena 9-10126 Torino, Italy.
Merlino
Chiara
C
Daniele
Roberta
R
Sinesi
Franca
F
Fumagalli
Mara
M
Ponzi
Alessandro Negro
AN
Cavallo
Rossana
R
D002478:Cells, Cultured; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D020031:Epstein-Barr Virus Infections / Q000097:blood* / Q000175:diagnosis; D005260:Female; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral