Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids.
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 10(6) viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 micro g) 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
Francesco
F
Unit of Human Virology, DIBIT, San Raffele Scientific Institute, 20132 Milan, Italy.
Locatelli
Giuseppe
G
Sarmati
Loredana
L
Piergiovanni
Sara
S
Veglia
Fabrizio
F
Andreoni
Massimo
M
Buttò
Stefano
S
Ensoli
Barbara
B
Lusso
Paolo
P
Malnati
Mauro S
MS
D017088:AIDS-Related Opportunistic Infections / Q000821:virology; D001826:Body Fluids / Q000821:virology*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D006566:Herpesviridae Infections / Q000821:virology; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D012514:Sarcoma, Kaposi / Q000821:virology; D012680:Sensitivity and Specificity; D019914:Taq Polymerase; D019562:Viral Load
D017931:DNA Primers; D004279:DNA, Viral; D019914:Taq Polymerase