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Calibrated Real-Time PCR Assay for Quantitation of Human Herpesvirus 8 DNA in Biological Fluids

Francesco Broccolo,¹ Giuseppe Locatelli,^1, Loredana Sarmati,² Sara Piergiovanni,¹ Fabrizio Veglia,³ Massimo Andreoni,² Stefano Buttò,⁴ Barbara Ensoli,⁴ Paolo Lusso,¹ and Mauro S. Malnati^1*

Unit of Human Virology, DIBIT, San Raffele Scientific Institute, 20132 Milan,¹ Department of Public Health and Cellular Biology, University "Tor Vergata," 00133 Rome,² I.S.I. Foundation Villa Gualino, 10133 Turin,³ Laboratory of Virology, Istituto Superiore di Sanità, 00161 Rome, Italy⁴

Received 4 March 2002/ Returned for modification 13 May 2002/ Accepted 13 September 2002

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.

Present address: Pharmacology Dept. Gene Expression Unit, Pharmacia, 20014 Nerviano, Milan, Italy.

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