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Journal of Clinical Microbiology, May 2000, p. 1981-1983, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Sensitive Method for Detection of Human
Herpesviruses 6 and 7 in Saliva Collected in Field Studies
Danielle M.
Zerr,1,*
Meei-Li
Huang,2
Lawrence
Corey,2,3
Matthew
Erickson,3
Heather L.
Parker,3 and
Lisa M.
Frenkel1,2
Departments of
Pediatrics1 and Laboratory
Medicine,2 University of Washington, and
Department of Infectious Diseases, Fred Hutchinson Cancer
Research Center,3 Seattle, Washington
Received 27 May 1999/Returned for modification 25 August
1999/Accepted 4 November 1999
 |
ABSTRACT |
To facilitate studies of the epidemiology and natural history of
human herpesviruses 6 and 7 in infants, a practical method for
collecting and quantifying the DNA of these viruses was developed. Saliva was collected using small strips of filter paper, and virus was
detected using a real-time quantitative fluorescent-probe PCR assay.
The sensitivity and specificity of this method even after prolonged
drying of the specimens compared favorably to those of our traditional
method of collecting and assaying saliva.
| |
TEXT |
Human herpesvirus 6 (HHV-6) and
human herpesvirus 7 (HHV-7) are almost universally acquired by 2 to 3 years of age (7, 8, 14, 16). Salivary viral shedding appears
to begin within days of primary infection (2, 19) and
persists in most individuals (6, 7). Thus, the detection of
virus in the saliva provides a noninvasive means of recognizing HHV-6
and HHV-7 infections, and a new onset of salivary shedding can be used
to detect recent acquisition of these viruses. We sought to develop a
simple, reliable method for detecting HHV-6 and HHV-7 in the saliva of infants.
Small commercially available filter paper strips have been utilized as
specimen collection devices for the detection of both viral antigens
and antibodies in local secretions (3, 4, 20; P. Reighelderfer, R. Coombs, D. Wright, D. Burns, and A. Kovacs, Abstr.
38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. I-251,
1998). We evaluated the use of these filter paper strips for the
collection of saliva and the detection of HHV-6 and HHV-7 DNAs.
Experimental design and collection of saliva.
Sno strips
(Chauvin Pharmaceuticals Ltd., Essex, England) are strips (60 by 6 mm)
of sterile filter paper designed to quantify tear flow. Approximately
12.5 µl of fluid has been shown to saturate the filter paper (data
not shown). Four different experiments were completed to evaluate both
the sensitivity of Sno strips as a means of collecting saliva for HHV-6
and HHV-7 PCR detection and the effects of environmental conditions on
this assay.
First, the detection of HHV-6 by PCR was used to compare saliva
collected by Sno strips and saliva collected by expectoration into a
cup (7). Saliva was collected from 33 healthy adults both by
placement of five Sno strips in the mouth until the distal ends were
saturated and by expectoration of approximately 0.4 ml of saliva into a
sterile cup. Specimens were stored at 
20°C until the DNA was
extracted with phenol-chloroform, amplified in a Perkin-Elmer 9600 thermocycler, and assayed by liquid hybridization (method 1)
(7). Second, using method 1, PCR to detect HHV-7 was
performed on the remaining extracted DNA from the saliva collected by
Sno strips. Third, duplicate sets of five Sno strips were collected from 10 individuals to compare method 1 with a more automated and
quantitative method that uses a Perkin-Elmer 7700 automatic thermocycler (method 2). For method 2, DNA was extracted using Qiagen
columns (QIAmp Blood Kits; Qiagen Inc., Valencia, Calif.), and PCR was
performed using a real-time quantitative fluorescent-probe PCR assay
(Perkin-Elmer Applied Biosystems, Foster City, Calif.). Finally, four
additional samples of five Sno strips each were obtained from seven
individuals. One sample from each individual was frozen immediately,
while the rest were allowed to air dry for 1 day, 1 week, and 2 weeks
before storage at 20°C to compare what effect prolonged drying
might have on our ability to detect HHV-6 and HHV-7 viral DNAs. These
samples were analyzed using method 2.
DNA extraction and PCR methods. (ii) Method 1.
Method 1, utilizing phenol-chloroform DNA extraction and liquid hybridization,
was performed as previously reported (7). Expectorated
saliva (0.4 ml) or the distal portions of five Sno strips cut at the
shoulder (60 µl) were treated overnight with proteinase K. DNA was
extracted using phenol-chloroform (7). The HHV-6 and HHV-7
DNAs from 12.5 µl of saliva from Sno strip specimens and 20 µl of
saliva from expectorated specimens were then amplified by PCR
(Perkin-Elmer 9600 thermocycler). The primer pair 5R and the probe 5R-P
were used for strain common detection of HHV-6 (7). The
HHV-7 primer sequences HHV7-1 (5' CGG CGT TTT ACT CGG AAC TCC T 3') and
HHV7-2 (5' TCC CCA TAA CAA ATG TGC CAT AAG A 3') amplified a 116-bp
portion of the major capsid protein. The probe HHV7-p1 (CAG ATT TTG TCC
AAC GCC CTA TC), end labeled with 32P, was used to detect
the amplicon by liquid hybridization and autoradiography
(7). These primers and probes have been found to reliably
detect 10 copies of purified HHV-6 or HHV-7 DNA and are specific when
tested with herpes simplex virus types 1 and 2, cytomegalovirus,
Epstein-Barr virus, varicella-zoster virus, and HHV-6 (for HHV-7
primers) (data not shown), HHV-7 (for HHV-6 primers) (7),
and HHV-8 (data not shown) target DNAs.
Negative controls included HSB-2 cultured T cells coprocessed with
every five study specimens, and at least one sample with
all reaction
components, except input DNA, was included in every
experiment.
Positive controls were also included in every experiment.
Fifty copies
of the internal control, the HHV-6 or HHV-7 amplicon
with 21 bp of the
probe sequence replaced by unrelated
Drosophila DNA (fly
control) (
5), were added to each PCR mixture prior
to
amplification. In addition, every experiment included a dilution
series
of cloned amplicon DNA that consisted of 10
1,
10
2, 10
3, and 10
4 copies.
(ii) Method 2.
Method 2 utilized Qiagen columns to isolate DNA
from Sno strips. The saturated ends from five Sno strips in 400 µl of
ATL tissue lysis buffer (Qiagen) with 800 µg of proteinase K were incubated overnight at 55°C. On the following day, 400 µl of AL lysis buffer (Qiagen) was added. After incubation at 70°C for 10 min,
420 µl of 100% ethanol was added. The mixture, including the strips,
was then loaded into the columns and centrifuged at 6,000 × g for 2 min. The DNA was eluted with 100 µl of 10 mM Tris.
Real-time PCR, a sensitive and reproducible method for the detection of
viral DNA (
17), was used to detect HHV-6 and HHV-7
DNAs.
Real-time PCR uses the 5'
3' exonuclease activity of
Taq polymerase to digest an internal probe labeled with two fluorescent
dyes, the reporter and the quencher (
10). When the probe is
intact, the reporter and quencher dyes undergo fluorescent resonance
energy transfer, thereby suppressing the fluorescence of the reporter
dye (
9). When target DNA is present, upon primer elongation,
the probe is cleaved by the 5'
3' exonuclease activity of
Taq polymerase. The reporter dye is then no longer
physically attached
to the quencher dye on the probe, and fluorescent
resonance energy
transfer is interrupted. This results in an increase
of reporter
dye fluorescence that is proportional to the amount of PCR
product
accumulated. DNA copy number is determined by calculation of
the
number of PCR cycles necessary for a standard curve of known
amounts
of DNA (10
4, 10
3, 10
2, and
10
1) to cross a fluorescent threshold and then
interpolation of the
unknowns. The threshold is set at the beginning of
the exponential
phase for the amplification being
run.
Each 50 µl of PCR mixture contained 20 µl of purified solution
containing the specimen DNA, 830 nM primers, 100 nM probe,
8%
glycerol, 60 nM internal passive control (6-carboxy-x-rhodamine
conjugated to the 5' end of 5'-GATTAG-3'), 5 mM
MgCl
2, 200 µM
each deoxynucleoside triphosphate (dNTP)
(except for 400 µM dUTP),
2.5 U of AmpliTaq polymerase
(Perkin-Elmer), 1.25 U of Taqstart
Taq polymerase antibody
(Clontech Inc., Palo Alto, Calif.), 0.05
U of
uracil-
N-glycosylase (UNG), and 50 copies of internal
control
DNA. The 5R probe for the HHV-6 amplicon was labeled with the
reporter dye, 6-carboxyfluorescein (FAM), on the 5' end and the
quencher dye, 6-carboxytetramethylrhodamine (TAMRA), on the 3'
end. The
forward primer for HHV-7 real-time PCR was 5'-TTT CCT
GTG ACA AAA GAA
GCA GTT A, and the reverse primer was 5'-ATC CCA
CAC GCT TTA CGG G. The
sequence and labels of the HHV-7 probe
were 5'-FAM-TTC CTG CGC AAT AAA
GTG AAA ACT GTT AGC ATT-3'-TAMRA.
The internal control fly probe was
labeled with the reporter 6-carboxytetramethylrhodamine
on the 5' end
and TAMRA on the 14th base (A). A minor group binding
protein (Epoch
Inc., Bothell, Wash.) was added to the 3' end of
the fly probe to
increase the melting temperature and optimize
its performance for
real-time PCR. With a Perkin-Elmer 7700 automatic
thermocycler, the PCR
cycling temperatures were as follows: after
2 min of incubation at
50°C followed by 2 min at 95°C, the samples
were subjected to 45 cycles of 95°C for 20 s followed by 60°C
for 1 min. A true
FAM-negative PCR must have a positive 6-carboxytetramethylrhodamine
signal; otherwise, the reaction is interpreted as
inhibited.
Saliva from 31 of 33 adults (94%; 95% confidence interval [CI], 80 to 98%) had detectable HHV-6 DNA. The two individuals who
did not have
HHV-6 detected in their saliva did not have HHV-6-specific
antibody
found by Western blot testing and were considered non-HHV-6
infected. A
comparison of HHV-6 detection in specimens collected
from the infected
individuals by expectoration and by Sno strips
revealed six specimens
that were discordant: three that were negative
by Sno strip and
positive by expectoration and three that were
positive by Sno strip but
not by expectoration (the PCR was inhibited).
Thus, the Sno strip and
expectoration methods were 90% sensitive
(28 positive of the 31 positive by either method) (95% CI, 75
to 96%).
Of the available Sno strip specimens, 28 of 29 (97%; 95% CI, 83 to
100%) had amplified HHV-7 DNA. HHV-7 serologic testing
of the
individual with no HHV-7 in his saliva was not done, and
the
expectorated specimens were not
tested.
A comparison of phenol-chloroform DNA extraction followed by
conventional PCR and liquid hybridization (method 1) to DNA extraction
by Qiagen columns followed by real-time PCR (method 2) revealed
that
the latter method had a sensitivity similar to that of the
former in
detecting HHV-6 DNA from specimens collected by Sno
strips. Overall, 9 of 10 specimens (90%) analyzed by conventional
PCR and liquid
hybridization were positive for HHV-6 DNA compared
to 10 of 10 specimens (100%) analyzed by real-time PCR. The DNA
copy number, as
determined by real-time PCR, ranged from 463 to
29,683 copies/ml. The
sample with the lowest copy number, 463
copies/ml, was the sample that
was found negative by method
1.
There did not appear to be a consistent difference in the detection of
HHV-6 or HHV-7 DNA from samples that were frozen immediately
or allowed
to dry for 1, 7, or 14 days at room temperature prior
to freezing and
then were tested. The mean copy numbers between
sample groups dried for
different amounts of time were not significantly
different (data not
shown). Interperson variability was high;
however, viral copy
number did not vary much within an individual's
samples (Fig.
1).
View larger version (38K):
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|
FIG. 1.
Quantities of salivary HHV-6 and HHV-7 DNAs detected in
specimens collected by Sno strips and allowed to dry for different
lengths of time prior to assay. (A) Saliva was collected from six
different individuals and allowed to dry for different time periods
prior to PCR. The graph depicts the log-transformed HHV-6 DNA copies
per milliliter of saliva at the different time points for the six
subjects. (B) Saliva was collected from six different individuals and
allowed to dry for different time periods prior to PCR. The graph
depicts the log transformed HHV-7 DNA copies per milliliter of saliva
at the different time points for the six subjects.
|
|
Sno strips appear to provide a simple, noninvasive, yet sensitive means
of collecting saliva in the field for the detection
of HHV-6 and HHV-7
DNAs by PCR. Overall, 90% of HHV-6-infected
adults had viral DNA
detected in their expectorated saliva, and
90% had DNA detected in
saliva collected by Sno strips. Previous
studies that have assayed a
larger volume of adult saliva (20
to 30 µl) by PCR have reported
frequent (90 to 100%) detection
of HHV-6 and HHV-7 DNAs (
1,
7,
11,
12,
15). Other
studies, in which saliva was collected using
devices such as cotton
swabs and throat swabs, have reported a lower
and variable frequency
of detection of HHV-6 (3 to 67%) (
13,
18). Although these
studies did not report the actual volume of
saliva assayed, it
may be that smaller volumes of saliva were used.
Relatively less
specimen was assayed when collected by Sno strips (10 to 15 µl)
than by expectoration (20 µl). Autoradiographic images
produced
from the Sno strip specimens were less intense than those
produced
from the expectorated specimens (Fig.
2), indicating that relatively
less HHV-6
DNA was amplified from the specimens collected by Sno
strips.
Furthermore, compared to the results for other subjects,
there was
relatively little HHV-6 DNA in the samples of the three
individuals
whose expectorated saliva was HHV-6 DNA positive and
Sno strip saliva
negative. This result suggests that the limit
of detection had been
reached for the individuals with relatively
lower viral DNA levels in
their saliva.
View larger version (57K):
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|
FIG. 2.
Autoradiogram of patient specimens (lanes 1 to 3 and 5 to 7) and a negative control (lane 4). Lanes: A, amplicons from
specimens collected by Sno strips; B, those collected by expectoration.
Lanes A have a relatively weaker signal or no signal (patient 6)
compared to their paired lanes B. Patient 6 appears to have less viral
DNA in her saliva than patients 1, 2, 5, and 7. HHV-6 DNA collected in
10 to 15 µl of saliva by Sno strips appears to be the limit of
detection by method 1.
|
|
Increasing the volume of saliva above the 10 to 15 µl that can be
wicked onto Sno strips would not necessarily increase the
overall
sensitivity of the test. Inhibitors of PCR became evident
in the saliva
of three individuals when more saliva (20 µl) was
assayed from
expectorated specimens. It is not evident whether
the lack of PCR
inhibition for the specimens collected by Sno
strips was due to the
smaller specimen volume or to the removal
of inhibitors by the filter
paper.
Method 2 may offer a slight increase in sensitivity over method 1. The
1 of 10 Sno strip specimens that tested negative for
HHV-6 when
phenol-chloroform was used to extract the DNA, followed
by routine PCR
(method 1), had a low copy number of HHV-6 when
DNA was isolated with
Qiagen columns and quantified by real-time
PCR (method 2). DNA
extraction with Qiagen columns followed by
real-time PCR offered
several clear advantages, including rapidity
(it required approximately
half the time to process specimens)
and objective quantification of the
DNA copy number, potentially
allowing for a better understanding of
viral dynamics. In addition,
pertinent to field studies, drying saliva
collected with Sno strips
for up to 2 weeks did not appear to
significantly affect the isolation
of viral
DNA.
In summary, Sno strips for saliva collection, Qiagen column isolation
of DNA, and fluorescent, real-time PCR provide a convenient
and
sensitive method for the study of HHV-6 and HHV-7 DNA
shedding.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Children's
Hospital and Regional Medical Center, CH-32, 4800 Sand Point Way, N.E., Seattle, WA 98105. Phone: (206) 528-5086. Fax: (206) 527-3897. E-mail:
zerr{at}u.washington.edu.
| |
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Journal of Clinical Microbiology, May 2000, p. 1981-1983, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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