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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,
Alberto
Gobbi,1,
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.

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

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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