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Journal of Clinical Microbiology, August 2001, p. 3020-3021, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.3020-3021.2001
LETTERS TO THE EDITOR
LightCycler-Based Quantitative PCR for Rapid Detection of
Human Herpesvirus 6 DNA in Clinical Material
 |
LETTER |
Locatelli et al. (6) reported on the quantitative
detection of human herpesvirus 6 (HHV-6) in plasma and cell suspensions by TaqMan-based PCR. We wish to add our experience. We have developed a
rapid (<30-min) assay for the simultaneous detection, quantification, and differentiation of HHV-6 variants A and B using a LightCycler (LC;
Bio/Gene Ltd., Kimbolton, England). The instrument performs real-time
PCR, combining rapid cycling with fluorescence-based identification of
PCR products in glass reaction capillaries (1).
We designed primers and a 5'-Cy5-labeled biprobe (2) to
target a 119-bp fragment of the large tegument protein gene. Detection of PCR product occurs when the biprobe hybridizes to the amplicon, leading to an increase in fluorescence resonance energy transfer between the double-stranded DNA fluorophore, SYBR green 1 and Cy5
(2, 5). The biprobe was designed to be complementary to
the variant B sequence and to detect a 2-bp polymorphism between the
variants (Table 1). Using
PCR-directed mutagenesis, a
plasmid-cloned quantitative standard (QS) was generated, incorporating
a single-nucleotide change in the same region with respect to the
variant B sequence, which could also be detected by the biprobe (Table
1). Simultaneous identification of the three targets with the biprobe
was achieved by melting curve analysis (1, 5), with
variants A and B and the QS yielding probe and product melting
temperatures of 52, 62, and 54°C, respectively (Fig. 1).
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FIG. 1.
Melting curve graph showing differentiation of HHV-6
variants A and B and the QS. Melting peaks were derived by plotting the
negative derivative of fluorescence ( dF/dT) with respect to
temperature.
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|
We routinely receive surveillance samples from immunocompromised
patients (including severe combined immune deficiency syndrome patients
and solid-organ and bone marrow transplant recipients) and have
evaluated the LC assay using not only plasma but also EDTA whole-blood,
serum, bone marrow, tissue, and respiratory samples, all extracted
using a simple commercially available method (QIAamp DNA minikit;
Qiagen, West Sussex, United Kingdom). To date, we have tested 129 samples in parallel with an in-house modification of a
solid-block-based nested PCR (9). Forty samples have
tested positive by both assays. Using the LC assay, all positive samples were identified as variant B, which has been shown to be the
predominant variant in transplant recipients (3, 4, 7, 8)
and the viral loads (ranging from 5 × 103 to
2.55 × 107 copies/ml) proved to be reproducible
(±0.5 log10 units).
Our assay offers rapid detection (<30 min, excluding DNA extraction),
quantitation, and typing of HHV-6 DNA in a closed-tube format which
minimizes the risk of carryover contamination. The assay is sensitive
(detection limit, 
10 copies), with a 6-log10 unit dynamic
range (10 to 
106 copies), is applicable to a wide range
of sample types, and compared well with nested PCR.
HHV-6 is an emerging pathogen and has been associated with severe
disease in the immunocompromised (3, 4, 7, 8). We concur
with Locatelli et al. (6) that determination of viral load
may be a useful indicator of active infection. The availability of
quantitative PCR methods means that results are available in a
clinically helpful time-frame (2 h by LC versus 2 days by nested PCR),
which should assist with implementing timely therapeutic intervention
and assessing response to treatment. In addition, use of this LC assay
may help to elucidate the epidemiology of the two variants and their
role in disease.
| |
FOOTNOTES |
*
Phone: 44 191 2261074
Fax: 44 191 2260365
E-mail: newakear{at}north.phls.nhs.uk
Ed. Note: The authors of the published
article declined to respond.
| |
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| | | | |
Angela M. Kearns*
Andrew J. L. Turner
Clive E. Taylor
Philip W. George
Roger Freeman
Newcastle Public Health Laboratory
Newcastle General Hospital, Westgate Road
Newcastle upon Tyne, NE4 6BE, United
Kingdom
|
| | | | |
Andrew R. Gennery
Paediatric Immunology and Infectious Diseases Unit
Newcastle General Hospital
Newcastle upon Tyne, NE4 6BE, United
Kingdom
|
Journal of Clinical Microbiology, August 2001, p. 3020-3021, Vol. 39, No. 8
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.8.3020-3021.2001
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