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Journal of Clinical Microbiology, November 2000, p. 4042-4048, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Real-Time Quantitative PCR for Human Herpesvirus 6 DNA

Giuseppe Locatelli,1 Fabio Santoro,1 Fabrizio Veglia,2,dagger Alberto Gobbi,1,Dagger Paolo Lusso,1,3 and Mauro S. Malnati1,*

Unit of Human Virology1 and Unit of Biostatistics,2 DIBIT, San Raffaele Scientific Institute, 20132 Milan, and Department of Clinical and Experimental Medicine, University of Bologna, 40138 Bologna,3 Italy

Received 23 March 2000/Returned for modification 5 June 2000/Accepted 28 July 2000

The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5' nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 106 viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.

* Corresponding author. Mailing address: Unit of Human Virology, Via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-2643-4903. Fax: 39-02-2643-4905. E-mail: malnati.mauro{at}hsr.it.

dagger Present address: I.S.I. Foundation, 10133 Turin, Italy.

Dagger Present address: Department of Experimental Oncology, European Institute of Oncology, 20142 Milan, Italy.

Journal of Clinical Microbiology, November 2000, p. 4042-4048, Vol. 38, No. 11
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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