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

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 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.

FIG. 1.

Reference curves of the HHV-8 TaqMan assay obtained in the presence and absence of irrelevant human genomic DNA. Serial dilutions (from 10¹ to 10⁶ HHV-8 genome equivalents) of the HHV-8 template run in the presence (closed circles) or absence (open triangle) of 1 μg of human genomic DNA derived from normal blood donor peripheral blood mononuclear cells. The equations resulting from the regression curves obtained by plotting the C_t values (y axis) against the indicated amount of DNA inputs (x axis) were the following: y = 39.226 − 3.3347 log (x), r² = 0.9978; and y = 39.667 − 3.4 log (x), r² = 0.9986 for the curve of the pVU56 construct diluted in water or in 1 μg of human DNA, respectively.

FIG. 2.

Comparison between the reference curves of the HHV-8 and calibrator real-time PCR assays. The number of the pVU56 (HHV-8) and pVU46 (calibrator) molecules is indicated on the x axis, and the cycle of threshold (Ct) is indicated on the y axis. Open triangle, TaqMan assay for HHV-8; closed circles, TaqMan assay for calibrator. The equations for the HHV-8 and the calibrator constructs were y = 39.052 − 3.333 log (x) and y = 39.084 − 3.340 log (x), respectively. The solid lines were obtained by linear regression analysis of the data (r² > 0.99; P < 0.001), and dotted lines indicate the 95% confidence intervals for the regression.