JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1995, p. 41–44 Vol. 33, No. 1
Copyright � 1995, American Society for Microbiology
Comparison of Three Methods for Extraction of Viral Nucleic
Acids from Blood Cultures
MARK J. ESPY, 1 ROBIN PATEL, 2 CARLOS V. PAYA, 2,3 AND THOMAS F. SMITH 1 *
Division of Clinical Microbiology, 1 Division of Infectious Diseases and Internal Medicine, 2
and Division of Experimental Pathology, 3 Mayo Clinic and
Foundation, Rochester, Minnesota 55905
Received 3 June 1994/Returned for modification 30 August 1994/Accepted 5 October 1994
Reliable nucleic acid extraction techniques for blood specimens are required for the sensitive detection of
viral DNA. Standardized procedures for processing blood specimens for the molecular detection of herpesviruses
(cytomegalovirus [CMV], herpes simplex virus, varicella-zoster virus, and Epstein-Barr virus [EBV])
have not been established. Three methods were used to extract DNA from blood specimens from healthy donors
and asymptomatic immunocompromised patients: (i) IsoQuick treatment of whole blood, (ii) extraction of the
peripheral blood leukocytes by lysis (lysis buffer and proteinase K), and (iii) extraction of peripheral blood
leukocytes with phenol-chloroform (sodium docecyl sulfate solution and proteinase K). All blood specimens
from 25 healthy blood donors were negative for CMV, herpes simplex virus and varicella-zoster virus nucleic
acid sequences, regardless of the extraction method, while three samples (12%) extracted by the lysis technique
were positive for EBV DNA. Of 25 blood samples from asymptomatic immunocompromised patients, CMV and
EBV each were detected in nine specimens by lysis extraction, four each by IsoQuick and four (CMV) and six
(EBV) by the phenol-chloroform method. Our results indicate that the lysis method is optimal for the detection
of CMV and EBV DNA sequences by PCR from the leukocytic fraction of blood specimens. DNA of these viruses
is frequently present in blood specimens from asymptomatic immunocompromised patients and occasionally
from healthy donors.
The importance of detecting herpesviruses (cytomegalovirus
[CMV], herpes simplex virus [HSV], varicella-zoster virus [VZV],
and Epstein-Barr virus [EBV]) in immunocompromised patients
can be difficult to interpret clinically. Detection of CMV
viremia in transplant recipients is the best viral marker to
predict symptomatic infection, including organ involvement (9,
15). Although CMV makes up over 97% of herpesvirus viremic
episodes in immunocompromised patients at our institution,
VZV and HSV are also recovered from blood of symptomatic
patients (2, 16). EBV, which causes lymphoproliferative disease
in immunocompromised hosts, is not recovered in cell
cultures used in routine diagnostic laboratories, thus making it
difficult to conclude the incidence and significance of EBV
viremia in these patients (22).
PCR allows detection of CMV, VZV, HSV, and EBV in
blood specimens. However, standardized procedures for processing
blood specimens for the molecular detection of these
herpesviruses remain to be established. Because of the clinical
importance of these viral infections in organ transplant and
AIDS patients in regard to antiviral prophylaxis, preemptive
treatment, and efficiency of therapy, we have compared three
different procedures for the optimal extraction of herpesvirus
DNA for subsequent PCR analysis: a commercial kit (Iso-
Quick), a lysis extraction procedure which uses a lysis buffer in
conjunction with proteinase K, and a phenol-chloroform extraction
followed by ethanol precipitation of the target nucleic
acid. With these results, we next analyzed the prevalence of
herpesvirus DNA in blood specimens from both healthy and
* Corresponding author. Mailing address: Division of Clinical Microbiology,
Mayo Clinic, 200 First St. S.W., Rochester, MN 55905.
MATERIALS AND METHODS
Clinical samples. Blood samples (5 ml) collected in EDTA-coated tubes were
obtained from 25 healthy blood donors, and 25 samples were collected from 21
consecutive organ transplant recipients who were monitored for the presence of
CMV viremia as part of routine protocols established in each transplant
program. Use of the samples for the evaluation described in this report was
approved by our institutional investigational research board.
Sample preparation. Whole blood was used for the IsoQuick (Microprobe
Corp., Bothell, Wash.) extraction kit (4). The leukocyte fraction of whole blood
was used for the lysis and phenol-chloroform extraction techniques. The
procedure used for leukocyte separation was as follows: 5 ml of whole blood was
layered over 7.5 ml of Histopaque 1119 (Sigma, St. Louis, Mo.), and the
combination was centrifuged at 700 � g for 25 min at 18 to 22�C. The top layer
(plasma) was discarded, and the second layer (leukocytes) was transferred to a
second centrifuge tube containing 5 ml of phosphate-buffered saline. The cells
were centrifuged at 700 � g for 10 min. The supernatant fraction was discarded,
and the cells were resuspended in 5 ml of medium (Eagle’s minimal essential
medium with 10% fetal calf serum). After centrifugation at 700 � g for 10 min,
the cells were resuspended in maintenance medium to a final volume of 2.0 ml
Inoculation of cell cultures. Leukocyte fractions obtained by Histopaque 1119
were suspended in 2 ml of Eagle’s minimal essential medium. Conventional tube
cell cultures (MRC-5; Viromed, Minneapolis, Minn.) were inoculated with 1 ml
each; three shell vial cell cultures received 0.3 ml each. Tube cell cultures were
incubated at 36�C on a roller drum for 14 days and examined three times a week
for the presence of cytopathic effects characteristic of CMV. Coverslips with
MRC-5 monolayers in shell vials were stained at 16 h postinoculation with
monoclonal antibody specific for the immediate early antigen of CMV (Bartels,
Issaquah, Wash.) by the indirect immunofluorescence test (11).
Serology. Antibodies to each of the herpesviruses were measured by the
anticomplement immunofluorescence test, using virus-infected substrate cells
obtained from Bion Enterprises, Park Ridge, Ill. (CMV, VZV, and HSV), or
Wampole, Cranbury, N.J. (EBV) (20).
Nucleic acid extraction. The IsoQuick extraction procedure was performed
according to the manufacturer’s instructions for processing whole-blood specimens.
Viral nucleic acid was extracted from leukocytes by lysis and phenolchloroform
methods (7, 8, 19) (Fig. 1).
Oligonucleotide primers and probes. Oligonucleotides specific for CMV,
VZV, HSV, and EBV were prepared with a DNA synthesizer (Applied Biosystems)
at the Mayo Clinic Molecular Core Facility, Rochester, Minn. In
addition, to ensure the quality of the nucleic acid extracted from the blood
sample, all specimens were tested for the presence of �-globin sequences by PCR
amplification (18). �-Globin sequences are found in all human tissues, and a
42 ESPY ET AL. J. CLIN. MICROBIOL.
FIG. 1. Procedures for extracting herpesvirus DNA from blood specimens.
The manufacturer’s instructions were followed for the IsoQuick procedure. TE,
Tris-EDTA; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline.
positive result after PCR indicated that the extracted material was of sufficient
quality for further PCR testing. Information regarding the primer and probe sets
is given in Table 1.
PCR amplification. The PCR reaction mixture contained the following: 200
�M (each) deoxyribonucleotide triphosphates, 10� buffer (500 mM KCl, 100
mM Tris-Cl [pH 8.3], 15 mM MgCl 2, 2.5 mg of bovine serum albumin per ml),
100 pmol each of the appropriate primer, 25 �g of isopsoralen per ml (IP-10;
HRI Associates, Concord, Calif.), 10% glycerol, and 1.25 U of Taq polymerase.
Each reaction tube received 45 �l of the reaction mixture, 2 drops of mineral oil,
and 5 �l of target DNA. A no-target control reaction tube received 50 �l of
reaction mixture only. Positive controls were included for each target tested. For
CMV, HSV, and VZV, American Type Culture Collection strains were inoculated
into cell cultures. DNA from each virus was extracted by the phenolchloroform
method and diluted such that the preparation produced the last
clearly visible band on gel electrophoresis. DNA extracted from a Burkitt’s
lymphoma cell line (Daudi cells) containing 118 to 224 copies of EBV per cell
and DNA from MRC-5 cells were positive control targets for the virus and for
the �-globulin gene, respectively (18, 21). Reactions were amplified in a DNA
thermal cycler (model 480; Perkin-Elmer Cetus), using the following protocol: 2
min of denaturation at 94�C for 1 cycle and then 1 min of denaturation at 94�C,
1 min of annealing at 60�C, and 1 min of primer extension at 72�C for 60 cycles
(7). After the cycling was completed, the tubes were placed in a UV transilluminator
(HRI-100; HRI Associates) for 15 min at 4�C to activate the IP-10 (8).
Identification of amplicon sequences. PCR products were visualized with UV
light as a single band by staining with ethidium bromide (10 mg/ml) after agarose
gel electrophoresis (3%:1.5% NuSieve [FMC, Rockland, Maine], 1.5% electrophoresis-grade
agarose [Bethesda Research Laboratories, Gaithersburg, Md.]).
The products were then transferred to a nylon membrane (Magnagraph; Fisher)
by Southern blotting. Membranes were prehybridized for 1hat42�C with a
hybridization solution obtained from Amersham (Arlington Heights, Ill.). The
probes were developed by first amplifying a sequence internal to each primer set.
These internal amplicons were then labeled for chemiluminescence with the
Enhanced Chemiluminescence kit from Amersham. The labeled probes were
added and allowed to hybridize to the membrane for 4 h. The membranes were
washed twice with 50 ml of wash buffer (6 M urea, 0.4% sodium dodecyl sulfate,
0.5� SSC [1� SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate]) at
42�C for 20 min and twice with 100 ml of 2� SSC at room temperature for 5 min.
The membranes were then placed in 30 ml of detection reagent provided in the
Enhanced Chemiluminescence detection kit for 1 min at room temperature and
then exposed to X-ray film for 1 h. The film was then developed by a Kodak
X-Omat X-ray film processor.
Healthy blood donor samples. Whole blood (IsoQuick extraction)
and leukocyte fractions (lysis and phenol-chloroform
methods) from healthy blood donors were negative for DNA
sequences by PCR and for detection of viral infectivity in cell
cultures for HSV, CMV, and VZV (Table 2). Of 25 serum
specimens from these donors, 18, 13, and 22 specimens contained
antibodies to HSV, CMV, and VZV, respectively. All
donors were seropositive to EBV. Blood specimens extracted
by IsoQuick and phenol-chloroform were also negative for
TABLE 1. Characteristics and nucleotide base sequences of primers and probes used to detect target nucleic acids
Organism Gene target
None �-Globin V00499 117–283 168 ACACAACTGTGTTCACTAGCA
HSV DNA polymerase M16321 2821–3110 291 TACATCGGCGTCATCTGCGGGG Primer
2855–3049 196 TCAAGGGCGTGGATCTGGTGCG Probe
VZV Gene 29 (major binding protein) 53730–54016 288 ATTATGGACTACGGCTTTTACT Primer
53761–53960 201 TTAGTCCGCGCGGCCATGAATC Probe
CMV Immediate early K01090 1067–1435 370 GGGTGCTGTCCTGCTATGTCTTA Primer
1117–1371 256 CGGCCTCTGATAACCAAGCCTG Probe
EBV Glycoprotein 220 V01555 90030–90268 240 GGCTGGTGTCACCTGTGTTA Primer
V01509 CCTTAGGAGGAACAAGTCCC Primer
V01554 90051–90243 194 ATTTTCACCACCTGTTGGGTGA Probe
VOL. 33, 1995 EXTRACTION OF VIRAL NUCLEIC ACIDS FROM BLOOD CULTURES 43
TABLE 2. Detection of CMV and EBV DNA sequences in blood
specimens from 25 immunocompromised patients a
No. of sequences detected by:
IsoQuickc Lysis Phenol-chloroform
4 9 4
4 9 6
a All specimens were negative for HSV and VZV.
b The gene target was immediate early antigen (370 bp).
c The gene target was glycoprotein 220 (240 bp).
EBV DNA; however, 3 of the 25 (12%) samples extracted by
the lysis technique were positive for this viral DNA target.
Immunocompromised patients. Collectively, of the 50 specimens
tested for CMV and EBV DNA, 8 specimens were
detected by both lysis and IsoQuick; however, 13 specimens
were positive for these viral nucleic acids by lysis but not by
IsoQuick (P � 0.01). Importantly, there were no specimens
that were positive by IsoQuick that were not detected by lysis.
Almost all serum samples were seropositive for both CMV
(23 of 25, 92%) and EBV (21 of 25, 84%). All PCR-positive
results from this patient population were from samples seropositive
to both viruses. All extraction techniques failed to
produce positive results for VZV and HSV DNA after PCR;
nevertheless, these primers consistently amplified these viral
targets in positive control samples and in routine assays performed
with cerebrospinal fluid and other specimens. DNA
specimens from healthy and immunocompromised patients
were positive for the �-globin gene, and control samples (positive
and negative) gave appropriate reactions for all targets
The clinical significance of a viral infection is enhanced by
the laboratory detection of the agent in the blood relative to
specimens obtained from other sources such as the respiratory
or gastrointestinal tract (10, 21, 23). Herpesvirus infections are
a recognized cause of morbidity and mortality in immunocompromised
patients, and PCR technology provides the potential
for the rapid and sensitive detection of these viruses, including
EBV. However, for the virology laboratory to be able to implement
PCR testing for these viruses, the optimal specimen
processing and extraction conditions must be defined for blood
specimens. In the past, preparation of blood specimens for
PCR was designed for use in human genetic analysis in which
the target copy was high (10 3 to 10 5 ) relative to the copies of
viral DNA expected with herpesvirus viremia (�10 1 to 10 2 ).
Therefore, at the present time, methods for the detection of
viremia by PCR have yet to be compared.
CMV and EBV replicate in the leukocyte fraction of blood
and are conventionally considered to be cell associated, with
relatively low levels of extracellular virus detectable in cell
cultures (22). Therefore, we combined the polymorphonuclear
and mononuclear leukocytic fractions and extracted DNA by
both the lysis and the phenol-chloroform procedures. These
two procedures involve an initial treatment with detergents to
solubilize cell components and with proteinase K to digest
histones that would otherwise remain strongly bound to DNA
(12). The phenol-chloroform extraction method would be
expected to reduce the concentration of residual protein and
membrane components which may potentially inhibit Taq
polymerase activity, whereas with the lysis method the leukocytic
fraction is boiled (but not extracted with organic solvents)
after proteolytic digestion. Nevertheless, with separated leukocytes,
we found that phenol-chloroform did not improve the
sensitivity of PCR to detect herpesvirus DNA. Comparatively,
the simpler lysis extraction with subsequent denaturation of
DNA by boiling provided maximum detection of both CMV
and EBV nucleic acid targets.
In our comparison, we included the IsoQuick kit, available
commercially, which is recommended for the extraction of
nucleic acids from blood samples. Since the procedural steps in
the lysis and phenol-chloroform methods are commonly not
uniform among laboratories, we included a technique which
could be easily standardized and accessible to diagnostic laboratories.
The IsoQuick technique utilizes the chaotropic
properties of guanidine thiocyanate, which both disrupts cellular
integrity and inhibits nucleases; the procedure is designed
to extract both DNA and RNA of purity and yield comparable
to that obtained by the phenol-chloroform method but without
the use of hazardous chemicals.
Our goal was to evaluate methods for detection of herpesvirus
DNA from either anticoagulated blood or the separated
leukocytes. However, it has recently been reported that serum
or plasma may be a suitable specimen for the detection of
CMV DNA by PCR (3). Although by culture analysis a small
proportion of extracellular virus is infectious, it is now being
realized that virus-specific DNA may be released in substantial
amounts into the extracellular serum or plasma phases of
blood. It would be fortuitous if the IsoQuick kit, because of its
commercial availability, had application to the rapid and
sensitive detection of herpesvirus DNA in these readily available
specimens in the clinical laboratory.
Because CMV and EBV replicate in peripheral blood leukocytes,
the detection of their nucleic acid in immunocompromised
patients was expected, as demonstrated previously (10).
In immunocompromised transplant recipients, we have previously
demonstrated that DNA sequences of CMV are routinely
present in both the mononuclear and cell-free fractions
(serum and plasma) of peripheral blood. In some cases, the
presence of CMV DNA does not correlate with symptomatic
infection or with culture documentation of viral replication (5,
17). The blood specimens in our study were selected on the
basis of their submission for viral culture and were collected as
part of a routine prospective protocol ongoing in organ
transplant recipients (liver , kidney , and bone marrow
), and none of the specimens studied correlate with clinical
or microbiological evidence of acute herpesvirus infection.
Conversely, blood specimens from 3 of 25 healthy blood
donors contained EBV but not viral sequences from other
herpesviruses (1). Similarly, in another study, EBV DNA was
detected in 15 of 16 seropositive healthy individuals (24).
These data indicate that the presence of EBV DNA in blood
specimens does not discriminate between infection in the
immunosuppressed and that in the immunologically normal
patient. Subsequent investigations should focus on the quantitative
levels of viral DNA present in blood specimens to
attempt to discriminate between symptomatic patients and
those without disease by using these markers.
Transition of research-based PCR to the routine clinical
laboratory requires strict adherence to technical steps designed
to reduce the likelihood of contamination of specimens containing
target nucleic acids with those samples free of virus.
For this purpose, we designated separate physical rooms for
specimen extraction and analysis of amplified products. In
addition, we use positive-displacement pipettors, together with
disposable gowns and gloves, as previously recommended (14).
Control for PCR contamination occurring prior to amplification
depends on following these recommendations; all extrac-
44 ESPY ET AL. J. CLIN. MICROBIOL.
tion procedures used in our evaluation (IsoQuick, lysis, and
phenol-chloroform) extraction would be expected to be of
equal risk regarding processing steps for potential sample-tosample
contamination. At least one target-free control for
every five patient samples was included in each test run. Most
importantly, post-PCR carryover contamination was controlled
by incorporation of isopsoralen into all reaction mixtures (8).
Reliable nucleic acid extraction techniques for blood specimens
are required for the sensitive detection of CMV infections
in immunocompromised patients. We recommend the
lysis method performed in this study with leukocytic fractions
of blood. Early and sensitive detection of this marker in blood
specimens may allow specific preemptive therapy for patients
at risk for the development of systemic disease due to this virus
(6, 13, 25).
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