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 μg) 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 [10.1128/JCM.40.12.4652-4658.2002].

Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids

BROCCOLO, FRANCESCO
Primo
;
LOCATELLI, GIUSEPPE
Secondo
;
2002

Abstract

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 μg) 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
Articolo in rivista - Articolo scientifico
Microbiology (medical)
English
2002
40
12
4652
4658
none
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 [10.1128/JCM.40.12.4652-4658.2002].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/136587
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