Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 106 viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 Î¼g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.
Broccolo, F., Locatelli, G., Sarmati, L., Piergiovanni, S., Veglia, F., Andreoni, M., et al. (2002). Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids. JOURNAL OF CLINICAL MICROBIOLOGY, 40(12), 4652-4658.
|Tipologia:||Articolo su rivista|
|Citazione:||Broccolo, F., Locatelli, G., Sarmati, L., Piergiovanni, S., Veglia, F., Andreoni, M., et al. (2002). Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids. JOURNAL OF CLINICAL MICROBIOLOGY, 40(12), 4652-4658.|
|IF:||Con Impact Factor ISI|
|Settore Scientifico Disciplinare:||Settore MED/17 - Malattie Infettive|
|Revisione (peer review):||Sì, ma tipo non specificato|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1128/JCM.40.12.4652-4658.2002|
|Stato di pubblicazione:||Pubblicato|
|Data di pubblicazione:||2002|
|Titolo:||Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids|
|Autori:||Broccolo, F; Locatelli, G; Sarmati, L; Piergiovanni, S; Veglia, F; Andreoni, M; Butto, S; Ensoli, B; Lusso, P; Malnati, M.S|
|Appare nelle tipologie:||01 - Articolo su rivista|