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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
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ABSTRACT |
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.
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INTRODUCTION |
Human herpesvirus 6 (HHV-6) is a
ubiquitous infectious agent isolated for the first time in 1986 from
patients with lymphoproliferative disorders (39).
Seroepidemiological surveys have demonstrated that HHV-6 is highly
prevalent in the human population (30), with primary
infection occurring between 6 months and 2 years of age (6, 8,
36). Primary infection is responsible for a febrile and eruptive
disease of infancy, exanthem subitum (48), occasionally
complicated by meningitis and meningoencephalitis (26, 33),
fulminant or chronic hepatitis (5, 47), and idiophatic
thrombocytopenic purpura (27). In immunocompetent adults, an
HHV-6 etiology has been suggested for several pathological entities
such as encephalitis (35; M. Ikusaka, K. Ota, Y. Honma, K. Shibata, S. Uchiyama, and M. Iwata, [Letter, Intern. Med.
36:157, 1997]), fulminant hepatitis (43)
Epstein-Barr virus-negative infectious mononucleosis (4,
45), chronic fatigue syndrome (9, 16, 37), and, more
recently, multiple sclerosis (2, 3, 11, 29, 44). A large
body of evidence suggests that HHV-6 may act as an opportunistic
agent in patients with immunodeficiencies, particularly those who have
undergone bone marrow or organ transplantation (10, 15, 18, 19,
42) and human immunodeficiency virus-infected individuals
(1, 17, 20, 28, 41). Moreover, a role of HHV-6 as a
cofactor in the progression of human immunodeficiency virus infection
toward full-blown AIDS has been proposed (30).
Despite an increasing number of clinical reports, however, the link
between HHV-6 and human diseases other than exanthem subitum has not
been definitively proved. Carefully controlled longitudinal studies are
still missing, mostly due to the lack of reliable markers of active
HHV-6 infection. Indeed, the serological determination of anti-HHV-6
immunoglobulin G (IgG) antibodies has only a limited value due to the
high prevalence of HHV-6 infection in the human population
(8) and the antigenic cross-reactivity with other beta-herpesviruses, such as human cytomegalovirus and HHV-7
(7, 25). Although the determination of titers of
anti-HHV-6 IgM antibodies that are either broadly reactive
(46) or that specifically recognize the p41 viral antigen
(1) may identify active or reactivated infection, the
specificity and sensitivity of such a determination are not optimal
since up to 5% of healthy adults may be positive, and conversely, many
culture-positive children fail to show detectable IgM titers (36,
38). Similar to IgG antibody titer determination, qualitative PCR
assays for detection of viral DNA in peripheral blood leukocytes cannot
discriminate between a latent infection, present in the vast majority
of healthy individuals, and an active one (30). Only
quantitative reverse transcriptase-based PCR assays (34)
with blood cells or quantitative PCR techniques applied to the
detection of free HHV-6 DNA in biological fluids may represent reliable
assays for tracking of an active HHV-6 infection. Nevertheless, the
routine application of these assays in large epidemiological surveys
has been hampered by their inherent technical difficulties and
labor-intensive nature.
Here, we describe the development of a new HHV-6 DNA-based PCR assay
for reliable estimation of the HHV-6 load in plasma and cell
suspensions. This assay, based on real-time quantitative PCR
(24), combines in a single step a PCR amplification reaction with the detection and computer-assisted quantification of the amplified product, thus resulting in a reliable and practical PCR
system well suited for the screening of large numbers of clinical samples.
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MATERIALS AND METHODS |
Oligonucleotide primers and TaqMan probe.
Primers TAQ6E
(5'-CAAAGCCAAATTATCCAGAGCG-3') and TAQ6A
(5'-CGCTAGGTTGAGGATGATCGA-3'), which amplify a 133-bp
fragment of the highly conserved U67 open reading frame of HHV-6, were
selected by using Primer Express software (PE Biosystems, Foster City, Calif.) and were synthesized by Primm (Milan, Italy). A 25-bp oligonucleotide probe (5'-CACCAGACGTCACACCCGAAGGAAT-3')
complementary to an internal region 28 bp downstream of the
forward primer was similarly selected and synthesized with a reporter
dye, 6-carboxyfluorescein, and a quencher dye,
6-carboxytetramethylrhodamine, covalently linked to the 5' and 3' ends,
respectively (PE Biosystems, Warrington, United Kingdom).
Preparation of HHV-6 standard and competitor DNA template.
To obtain a construct to be used as a reference for the quantitation of
HHV-6, we extracted the DNA from the culture supernatant of peripheral
blood mononuclear cells (PBMCs) infected with HHV-6AGS and
amplified a fragment of 133 bp derived from the U67 region (Fig.
1) with a PCR mixture containing 100 mM
deoxynucleotides, 3 mM magnesium chloride, 1× TaqMan buffer A, primers
TAQ6E and TAQ6A (each at 300 nM), 0.625 U of AmpliTaq Gold, and 40%
(vol/vol) DNA template. The cycling profile consisted of a first
denaturation step of 15 min at 95°C; a second step of 26 cycles with
denaturation for 20 s at 95°C, annealing for 30 s at
58°C, and extension for 30 s at 72°C; and a final extension
step of 10 min at 72°C, which closed the amplification protocol. The
amplified 133-bp product was then cloned into the pCRII plasmid by
using the TA cloning kit (Invitrogen Corp, San Diego, Calif.) according
to the manufacturer's instructions.
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FIG. 1.
Schematic representation of the HHV-6 sequence with the
standard and competitor DNA constructs.
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A competitor construct was obtained by amplifying the HHV-6 reference
DNA molecule with the standard forward primer (TAQ6E)
and the reverse
primer 5'-CGCTAGGTTGAGGATGATCGAATTCCTTCGGGTGTGACGTCT-3'.
This primer combines the first 21 nucleotides of the reverse
primer
TAQ6A with 21 additional nucleotides complementary to the probe;
therefore, the amplification reaction yields a 96-bp fragment
that
corresponds to the standard fragment (133 bp) but that lacks
the 37 bp
that separate the probe from the reverse primer (Fig.
1). The inserts
in the standard (pVU44) and the competitor (pVU45)
constructs were
first fully sequenced with the ThermoSequenase
kit (Amersham Pharmacia
Biotech AB, Uppsala, Sweden); then, the
plasmids were expanded and
accurately quantitated by evaluation
with a
spectrophotometer.
PCR assay conditions.
The TaqMan reaction was performed in a
final volume of 25 µl containing 100 mM each dATP, dCTP, and dGTP;
200 mM dUTP; 5.5 mM magnesium chloride; 1× TaqMan buffer A; primers at
300 nM; probe at 50 nM; 0.625 U of AmpliTaq Gold; 0.25 U of
uracil-N-glycosylase; and 40% (vol/vol) DNA template. The
TaqMan PCR cycling conditions were 2 min of degradation of preamplified
templates at 50°C, followed by 15 min of denaturation at 95°C and
then 40 cycles of denaturation at 95°C for 15 s and annealing
and extension at 58°C for 60 s. The normalized fluorescent
signal (
Rn) generated by the degradation of
the hybridized probe was automatically calculated by a computer algorithm that normalizes the reporter emission signal first by dividing it by the emission of a control dye (ROX) present in the PCR
mixture and then by subtracting all the background signals generated in
the first 15 cycles of the PCR. The algorithm then calculates the
threshold cycle (Ct) at which each PCR
amplification reaches a Rn threshold value
(usually set at 10 times the standard deviation of the baseline signal)
that is inversely proportional to the log number of target copies
present in the sample (24).
Quantitative-competitive PCR (QC-PCR) was performed with a fixed input
of 1,000 copies of the synthetic competitor DNA molecule
of 96 bp
described above per reaction mixture. The reaction was
carried out in a
final volume of 50 µl, with 1× PCR buffer II
(PE Biosystems, Foster
City, Calif.), 5.5 mM magnesium chloride,
100 mM deoxynucleoside
triphosphates, 2.5 U of AmpliTaq Gold,
primers at 3 mM, and 20%
(vol/vol) DNA template. The thermal cycler
conditions were set as
follows: 2 min at 50°C, 15 min at 95°C,
and 45 cycles of 15 s
at 95°C and 1 min plus 8 s per cycle at
58°C. The
amplification products were stained with ethidium bromide
and were
analyzed by agarose gel electrophoresis. The intensity
ratio of the two
bands, corresponding to the DNA template and
the competitor DNA
amplicons, was determined by densitometric
analysis with Image Quant
software (Amersham Pharmacia Biotech
Inc., Piscataway, N.J.) The amount
of HHV-6 genome equivalents
present in the test samples was calculated
by interpolation on
a standard curve, obtained by plotting the value of
the template/competitor
ratio against the known initial copy number of
standard DNA templates
on a semilogarithmic scale (see Fig.
5).
The nested PCR assay was performed with two sets of primers, as
described previously (
40).
Sample preparation.
DNA was isolated from 100 µl of plasma
or culture supernatant or from 5 × 104 cells derived
from thymic grafts explanted from SCID-hu Thy/Liv mice inoculated with
HHV-6AGS (21). Extraction of DNA from biological fluids was performed with 0.5 ml of a lysis buffer containing 10 mM
Tris-HCl (pH 8), 5 mM EDTA, 0.5% (vol/vol) sodium dodecyl sulfate, and
0.1 µg of proteinase K per ml. DNA was extracted from the cells with
0.1 ml of a lysis solution containing 100 mM KCl, 10 mM Tris-HCl (pH
8.3), 2.5 mM MgCl2, 0.5% (vol/vol) Tween 20, 0.5%
(vol/vol) Nonidet P-40, and 0.5 µg of proteinase K per ml. In both
instances, lysis was followed by phenol-chloroform extraction and
high-salt isopropanol precipitation; the purified DNA was resuspended
in 100 µl of a 5 mM Tris-HCl-0.5 mM EDTA buffer and was stored at
20°C until use.
To control for potential cross-contamination during sample
manipulation, one negative control (plasma from a healthy volunteer
or
DNA extracted from uninfected thymic grafts) was included after
each
pair of test samples; the three different steps of the PCR
procedure
(DNA extraction, preparation of the PCR mixture, and
addition of the
DNA to the PCR mixture) were performed in three
physically separated
and dedicated
rooms.
Preparation of viral stocks.
PBMCs were obtained from
healthy donors by gradient centrifugation, activated in vitro with
purified phytohemagglutinin for 2 days, and infected with
HHV-6AGS or HHV-6BPL1 at a multiplicity of
infection of 0.1 (i.e., 1 infectious particle per 10 cells). Two hours
after infection, the cells were washed and incubated for several days
at 37°C in a humidified CO2 incubator. The culture supernatant was harvested after 7 to 10 days of infection, and virus
titration was performed by infecting triplicate cultures of
phytohemagglutinin-activated PBMCs with serial 10-fold dilutions of the
viral stock (31).
Statistical analysis.
Accuracy, defined as the level of
approximation of a measured value to a reference value taken as a
"gold standard," was estimated by computing the arithmetic
differences between the number of DNA copies evaluated by the two
assays and the theoretical copy number expected according to UV
spectroscopy. The significance of systematic biases was assessed by a
paired-sample Student t test and was adjusted by covariance
analysis, when needed.
Repeatability (i.e., the variability of a method when repeated measures
are taken with the same material in a single experiment)
and
reproducibility (i.e., the variability of a method when repeated
measures are taken in different experiments) were estimated by
computing the coefficient of variation (CV; the ratio between
the
standard deviation and the mean of repeated measurements)
under
different conditions. The significance of the differences
between
methods was assessed by the
F test.
The difference between reference curves was assessed by covariance
analysis. The Duncan method was used to account for multiple
tests.
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RESULTS |
Optimization of TaqMan assay conditions.
We first
achieved reproducible detection of three doses of reference DNA
(102, 104, and 106 copies/reaction
mixture) by using the standard PCR conditions suggested by the 7700 ABI
Prism sequence detector manufacturer (data not shown). Then, we
exploited the conditions of the TaqMan reaction to ensure both the
optimal kinetics of fluorescent signal accumulation and the earliest
measurement of the Ct values over a wide range
of known template DNA concentrations. As illustrated in Table
1, several parameters were optimized by
varying each of them in the context of the standard PCR conditions;
primer and probe concentrations were tested between 50 and 900 nM and between 10 and 200 nM, respectively, whereas the Mg2+
concentration was varied between 1.5 and 7 mM. The best amplification profiles were obtained with each primer at a concentration of 300 nM in
conjunction with Mg2+ at a concentration of 4 mM or higher.
A probe concentration of 50 nM was sufficient to ensure both the
overall best Ct values (Table 1) and an optimal
accumulation of the fluorescent signal (Rn);
the latter was roughly proportional to the initial probe concentration
(data not shown). The optimal AmpliTaq Gold activation time (Table 1)
and the annealing-extension temperature were also experimentally
determined; the highest sensitivity was achieved with an
activation time of 15 min and an annealing-extension temperature of
58°C (data not shown), 3°C degrees under the predicted primer melting temperature.
Validation of reference curve.
The 133-bp HHV-6 DNA fragment
amplified by primers TAQ6A-TAQ6E was cloned into the pCRII plasmid,
thus providing a reproducible source of standard DNA (Fig. 1). To
generate the reference curve, the plasmid DNA was accurately
quantitated by UV spectroscopy, and three distinct sets of 10-fold
dilutions were prepared starting from a DNA concentration of 0.5 µg/µl (equivalent to 1011 copies per µl) and ending
with a DNA concentration of 0.0005 fg/µl (equivalent to
10
1 copies per µl). The three series of samples were
then amplified by PCR in the same run of the 7700 ABI Prism sequence
detector. All the data collected were used to generate a log-linear
regression plot (Fig. 2A) that showed a
strong linear relationship (r = 0.99) between the log
of the starting copy number and the Ct values. By covariance analysis, no significant differences were found among the
plots generated by the three distinct sets of dilutions (F = 2.40 and P = 0.1 and
F = 1.1 and p = 0.33 for the intercept and the slope of the plot, respectively), with the contribution of the
dilution procedure to the overall error rate being not higher than 8%
(Fig. 2B).
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FIG. 2.
(A) Reference curve for TaqMan assay. The dotted lines
represent the 95% confidence limits of the assay; the equation of the
regression curve obtained by plotting the Ct
values (y axis) against the indicated amounts of DNA inputs
(x axis) is y = 38.534 3.495 log(x), with a value of fit (R) equal to 0.999. (B) Results obtained with three reference curves, constructed at
different times, to assess reproducibility. In all the resulting
equations [curve 1, y = 38.907 3.554 log(x); curve 2, y = 38.547 3.508 log(x); curve 3, y = 38.147 3.422 log (x)], both the slope coefficients and the intercept
values were very similar, and no significant difference was found by
covariance analysis.
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Comparative analysis of TaqMan assay with other PCR techniques. (i)
Assay sensitivity and dynamic range.
The sensitivity of the TaqMan
assay was assessed in parallel by a highly sensitive nested PCR
protocol (40); serial dilutions of the standard plasmid, as well as a
crude HHV-6AGS stock, were used as templates. All the
serial dilutions that yielded a detectable band by nested PCR
generated a well-defined amplification plot (Fig.
3). The TaqMan assay was characterized by
a very wide dynamic range, as it could discriminate between
100 and 106 HHV-6 genome equivalents in a
single reaction. The sensitivity of the two systems was similar, since
both required at least three replicates to reliably detect a
single-copy input (data not shown). Greater starting copy numbers were
measurable, but due to the premature detection of the fluorogenic
signal, sample quantitation was possible only by decreasing the number
of background measurements that allow threshold
Rn calculation.
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FIG. 3.
Comparison of assay sensitivity for HHV-6 DNA detection
between TaqMan and nested PCR assays. The results are for serial
10-fold dilutions of the reference DNA (from 101 and
106 copies/reaction mixture) and one negative control
sample, as detected by TaqMan assay amplification (A) or by nested PCR
on an ethidium bromide-stained 2% agarose gel (B). The symbols shown
at the top of the nested PCR bands identify the corresponding dilutions
of the template. For each dilution, the normalized fluorescence signal
(Rn) is plotted against the PCR cycle
number.
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(ii) Accuracy, repeatability, and reproducibility.
Next, we
measured the accuracy of the TaqMan method and compared it with that of
a newly developed QC-PCR assay which amplifies the same target sequence
(Fig. 4) (see Materials and Methods). As
shown in Fig. 5, in seven distinct experiments performed
by the TaqMan assay, the error observed with serial dilutions of the
pVU44 reference plasmid was minimal (4.9 to 1.5%) for DNA inputs above
100 copies/reaction mixture (102 to 106),
whereas it was greater for inputs below 100 copies/reaction (7 and 23%
for 100 and 101 copies/reaction, respectively).
Moreover, we did not observe a significant bias of under- or
overestimation even after adjusting for the different amounts of
starting copy input (P = 0.75 by covariance analysis).
By contrast, the degree of error measured in three independent
experiments by the QC-PCR technique was significantly higher (11 to
102%) (Fig. 5). Moreover, a significant (P = 0.002) and systematic overestimation bias (values 56% higher than the expected values) was observed. Finally, comparison of the accuracies of
the two assays showed a marked difference (P = 0.005)
for the quantitation of the standard.
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FIG. 4.
Standard curve for HHV-6 QC-PCR. A fixed amount
(103 copies) of the pVU45 calibrator plasmid was amplified
in the presence of increasing amounts of the pVU44 standard plasmid
(A). Following ethidium bromide staining the relative amounts of
amplified products were calculated by densitometric analysis, and
ratios between the coamplified products were plotted on a
semilogarithmic scale as a function of increasing amounts of the
reference DNA (B). The corresponding equation is reported in the upper
part of the figure.
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FIG. 5.
Accuracy error of the TaqMan and QC-PCR assays, computed
as the average of the measured value after subtraction of the actual
value. The error is reported as a function of the actual number of DNA
copies to be measured. By both methods, accuracy improved with an
increase in the amount of DNA, but the error was systematically lower
by the TaqMan assay. In addition, QC-PCR yielded a marked
overestimation of the number of copies at all levels.
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The repeatability and reproducibility of the TaqMan assay were assessed
by quantifying both the reference DNA molecule (Table
2) and viral DNA extracted from
HHV-6-infected tissues obtained
from SCID-hu Thy/Liv mice (Table
3) or
human plasma. A progressive
decrease of the CV (from 197 to 5%) was
seen with an increase
in the starting DNA amount in the intra-assay
evaluation of the
reference DNA. Similarly, the interassay CV decreased
sharply
(from 197 to 14% between 10
0 and 10
3
copies/reaction mixture) and then reached a plateau, with an
average CV
of 8% for all the remaining DNA concentrations (from
10
3
to 10
6 copies/reaction). No statistical difference was
found between
the intra- and the interassay CVs, confirming the
reliability
of the entire technical procedure over time. The CVs for
repeatability
and reproducibility of the QC-PCR method, measured with
the same
reference molecule, were significantly worse (decreases in the
CV from 124 to 25% for repeatability and from 274 to 40% for
reproducibility)
than those measured by the TaqMan assay.
Quantification of the viral load in five samples derived from
heterochimeric mice experimentally infected with HHV-6A
GS
showed
that the QC-PCR assay significantly overestimated
(
P = 0.0001)
the TaqMan assay measurements by an
average of 40% (Table
3).
The
repeatability was similar by both methods (CVs, 4 to 24% and
6 to 31%
for the TaqMan assay and the QC-PCR, respectively), whereas
for three
of five samples (samples 1 to 3) the reproducibility
of the TaqMan
assay was superior to that of the QC-PCR assay and
it was significantly
worse in only one case (sample 5).
To further verify the validity of the TaqMan assay for quantitation of
biological samples, a set of human plasma samples previously
assayed
for the presence of HHV-6 DNA by nested PCR was tested.
Three samples
were chosen because they occasionally tested positive
by nested PCR,
three were chosen because they were consistently
positive, albeit only
after the second round of amplification,
and the last three were chosen
because they were already positive
after the first round of
amplification. By the TaqMan assay, marked
differences in the viral
loads in the three sets of samples were
documented (range, 2 to 23,555 copies/ml); the repeatability and
reproducibility for each sample were
similar (intra-assay CV range,
12 to 135%; interassay CV range, 12 to
156%), with the CV values
being not significantly different from the
ones measured with
the DNA reference
standard.
Detection of HHV-6 subgroups.
Lastly, we studied the ability
of the TaqMan assay to quantitate the DNA loads of both HHV-6 subgroups
(subgroups A and B). Titrated culture supernatants collected from PBMCs
infected with HHV-6 strains GS (subgroup A) and PL1 (subgroup B)
containing either 0.1 or 10 50% tissue culture infective doses
(TCID50s) per µl were tested for the HHV-6 DNA load. As
shown in Fig. 6, similar amounts of HHV-6
DNA of both subgroups were detected, with a consistent ratio
(approximately 2 logs at the two dilutions tested) between the number
of viral genome equivalents measured and the number of
TCID50s used.
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FIG. 6.
Detection of HHV-6 subtypes by TaqMan assay.
HHV-6AGS (filled columns) and HHV-6B PL1
(shaded columns) genome equivalents measured in 1 ml of supernatant
derived from cultured PBMCs infected with 0.1 and 10 TCID50s, respectively.
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DISCUSSION |
The laboratory diagnosis of active HHV-6 infection is a complex
process that often requires the simultaneous assessment of multiple
parameters. The reasons for such complexity are intrinsic to the HHV-6
life cycle: the virus enters the body during primary infection, often
clinically manifested by exanthem subitum, followed by a variable
period of clinical latency, during which the virus persists in selected
anatomical sites from where it is probably never cleared. Subsequently,
either reactivation or reinfection may occur, particularly in
concomitance with episodes of immune dysregulation or deficiency; these
can be followed, again, by variable periods of latency. As a
consequence of this pattern of alternating latency and reactivation,
the diagnostic value of anti-HHV-6 IgG antibody titers is limited.
Similarly, qualitative DNA PCR with blood cells is unable to
distinguish the maintenance of the viral genome in latently infected
cells (cellular latency, clinically silent) from the presence of cells
actively producing HHV-6 (often clinically relevant). Furthermore,
quantitation of the viral load with blood cells (14, 15, 23,
40), although suggestive of actively replicating virus, does not
provide direct proof of active or reactivated infection. Even isolation
of virus from blood cells, even though it is generally successful
during episodes of active virus replication, cannot formally
distinguish between active and latent HHV-6 infection, as it requires
the in vitro stimulation of the patient's cells, thereby potentially reactivating HHV-6 from latency. By contrast, the identification of
cell-free viral products (either antigens or DNA) in body fluids can
substantiate the diagnosis of active HHV-6 infection. To date, one
HHV-6 antigen-capture test has been described (32), but its
suitability for clinical use remains uncertain. The most practical test
remains the detection of HHV-6 DNA by qualitative PCR techniques in
serum, plasma, or other body fluids (13, 41). However, the
assays developed to date present two major limitations: first, they are
not extremely sensitive (12, 14); second, they cannot establish if the viral DNA is indeed derived from circulating virions
or from latently infected cells accidentally damaged in vivo or ex vivo
during sample manipulation. This problem can be circumvented by
combining quantitative PCR techniques that measure the viral load and
the cellular DNA content in the corpuscular and the liquid fraction of
the same sample.
To reduce the marked variability of PCR results due to endpoint
quantitation of the amplified products, the currently available QC-PCR
assays must introduce a synthetic DNA competitor molecule that allows
normalization for intra- and interreaction variability and that
also reveals potential PCR artifacts due to Taq polymerase inhibitors, technical inaccuracies, etc. (13, 23, 40).
Unfortunately, this introduces a significant degree of complexity into
the PCR assay, which increases the time and attention required
for determination of the correct result for a sample, thereby strongly
limiting routine application. Conversely, the real-time detection of
PCR amplification products measures the target DNA during the
exponential phase of growth in the PCR, thus circumventing many
of the problems connected with quantification at the plateau stage.
This technological breakthrough combines a high level of accuracy and
reproducibility with an extreme sensitivity and detection range
simply by the use of an external reference curve.
Of importance, partial inhibition of the PCR, which may reduce the
quantitation accuracy, can be revealed by alterations of the TaqMan
assay detection curve in comparison with the amplification plots
generated by the standards (G. Locatelli and M. S. Malnati, unpublished data). Qualitative alterations of the reference DNA can also be easily identified by virtue of the high interassay reproducibilities of the standard curve parameters. The only
limitation of this assay, in comparison with QC-PCR, is represented by
its inability to recognize PCR artifacts due to a complete failure of
the PCR (false-negative results); conversely, the use of a specific
probe to detect the amplified DNA permits, as in the Southern blot
technique, limitation of the occurrence of false-positive results.
Indeed, with over 300 measurements for a reference DNA molecule, the direct comparison of the TaqMan and QC-PCR assays developed with the same DNA sequence demonstrated that the real-time technique is a far more practical and reliable method of DNA
quantitation. The design of our assay also guarantees the same level of
efficiency and reproducibility for the quantitation of both the A and
the B HHV-6 subgroups; furthermore, we showed that it is equally
applicable to both liquid and cellular fractions of biological samples.
In conclusion, the real-time HHV-6 DNA detection assay described herein
offers several advantages over the traditional QC-PCR method: first,
the time required for labor, costs, and sample handling time are
greatly reduced because a single PCR run without further
postamplification steps is sufficient to accurately quantitate the
target DNA; second, the absence of postamplification manipulation steps
greatly reduces the risk of intersample contamination; lastly, the
96-well format provides a high-throughput system that makes this assay
suitable for the large-scale evaluation of HHV-6 infection in human
pathology (29), as well as in experimental animal models (21, 22).
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ACKNOWLEDGMENTS |
This work was supported by the European Union Biomed 2 (grant P1951301).
We thank C. A. Stoddart and J. M. McCune for kindly providing
tissue samples from SCID-hu Thy/Liv mice and Stefania Laus for excellent editorial assistance.
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FOOTNOTES |
*
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.
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Journal of Clinical Microbiology, November 2000, p. 4042-4048, Vol. 38, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
