Detection of human parvovirus B19 DNA by using the polymerase chain reaction.
ABSTRACT The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699 nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a 699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR. PCR was 10(4) times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 10(7) times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19 infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22 serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid, ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when testing only a single serum sample.
Article: Human parvovirus B19Seminars in Pediatric Infections Diseases 04/1996; 7(2):89–96.
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JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 65-69
Copyright © 1990, American Society for Microbiology
Detection of Human Parvovirus B19 DNA by Using the
Polymerase Chain Reaction
WILLIAM C. KOCH* AND STUART P. ADLER
Division ofInfectious Disease, Department ofpediatrics,Children's Medical Center, Medical College of Virginia,
Richmond, Virginia 23298
Received 16 June 1989/Accepted 28 September 1989
The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in
sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699
nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a
699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after
Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot
blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR.
PCR was 104 times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 107
times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19
infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22
serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples
lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid,
ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human
parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when
testing only a single serum sample.
Human parvovirus B19 (B19) causes several syndromes,
including erythema infectiosum, chronic arthritis in adults,
aplastic crisis in patients with hemolytic anemias, fetal
death, and chronic anemia and neutropenia in immunocom-
promised patients (2, 3, 7, 10, 15, 17, 20, 22, 25). The virus
replicates only in erythroid precursor cells derived from
bone marrow (17, 19). In vitro culture systems producing
human parvovirus B19 have not been developed. Conse-
quently, diagnostic tests for this infection are not widely
available. The few laboratories that do these tests must rely
on antigen obtained from the serum of infected individuals
(4). In addition, we and others have observed chronic B19
infection of immunocompromised patients whose serum
lacked immunoglobulin G (IgG) and IgM to the virus (15, 16;
W. C. Koch and S. P. Adler, J. Pediatr., in press). Diagnosis
was made by detecting B19 DNA in their sera by dot blot
analysis. To circumvent these problems in diagnosis and
because the polymerase chain reaction (PCR) is sensitive
and specific for detecting several other viruses, we evaluated
the PCR for the detection ofhuman parvovirus B19 in serum
and other body fluids (1, 8, 13, 18, 23).
MATERIALS AND METHODS
Selection of sequences for primers and probes. B19 has a
single-stranded DNA approximately 5,400 nucleotides long
(12). The B19 genome encodes two capsid proteins, VP1 (84
kilodaltons) and VP2 (58 kilodaltons) and a nonstructural
protein (77 kilodaltons) (11). VP1 and VP2 share common
carboxy-terminal amino acids. VP2 composes 96% of the
viral capsid. We evaluated three sets ofprimers and probes:
one set located within VP1, one set within the nonstructural
gene, and one set within VP2 (Table 1). Primers and probes
were synthesized on a DNA synthesizer (model 380A;
Applied Biosystems, Foster City, Calif.) and purified by
high-pressure liquid chromatography before use.
Amplification. Amplification of target sequences occurred
in a 500-,ul polypropylene microfuge tube with a total reac-
tion volume of 100 ,ul. The reaction mixture contained 200
,uM each dATP, dCTP, dGTP, and dTTP; oligonucleotide
primers (each at 1 FjM); 50 mM KCl; 10 mM Tris (pH 8.3);
2.0 mM MgCl2; 0.01% gelatin; and 1 to 3 ,ul of sample. After
the mixture was heated to 94°C for 3 min, 2.0 U of Taq
polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) was
added. The mixtures were overlaid with 100 ,ul ofmineral oil,
and then thermal cycling was carried out in a programmable
heat block (Perkin-Elmer). Each cycle consisted of 2 min at
94°C, 2 min at 37°C, and 3 min at 72°C. An additional 7 min
was added at the end of the cycling to complete extension of
the primers. Thirty-five cycles were performed in 6 h.
Detection of amplified B19 sequences in reaction products.
After cycling, 10 ,uI of each amplified mixture was electro-
phoresed on a 4% agarose minigel (3% NuSieve, 1%
SeaKem; FMC Corp., Rockland, Maine). After gels were
stained with ethidium bromide, they were viewed under UV
light and photographed. Southern transfer and hybridization
with a B19-specific probe were performed by electrophoret-
ically transferring DNA fragments from the agarose gels to
nylon membranes (Nytran; Schleicher & Schuell Co.,
Keene, N.H.) in TAE buffer (10 mM Tris hydrochloride, 5
mM sodium acetate, 0.5 mM EDTA [pH 7.8]). Before
transfer, the gels were soaked in 0.2 N NaOH-0.5 M NaCI
for 30 min, followed by two washes of 10 min each in 500 ml
of4x TAE buffer and a final 10-min wash in 1x TAE buffer.
After the membrane was presoaked in 1 x TAE buffer for 5
min, the membrane and gel were assembled as a sandwich
between filter paper and placed in the middle slot of a
Trans-Blot tank (Bio-Rad Laboratories, Richmond, Calif.),
and 2.5 to 3.0 liters of lx TAE buffer was added. After
electrophoretic transfer overnight at 250 mA, the mem-
branes were baked at 80°C. The membranes were prehybrid-
ized at 42°C for at least 3 h in hybridization solution, which
contained the following: 6x SSPE (20x SSPE is 3.6M NaCl,
Vol. 28, No. 1
KOCH AND ADLER
TABLE 1. Location in the B19 genome and sequences of oligonucleotide primer pairs and probes
200 mM NaH2PO4 [pH 7.4], and 20 mM EDTA [pH 7.4]), 1%
sodium dodecyl sulfate (SDS), 10x Denhardt solution (0.2%
bovine serum albumin, 0.2% polyvinylpyrrolidone, and 0.2%
Ficoil), 20 ,ug oftRNA per ml, and 50 ,ug of sheared salmon
sperm DNA per ml. Then 0.2 ,ug of each probe was end
labeled to a specific activity of at least 2 x
gamma-labeled [32P]ATP by using T4 kinase (Bethesda Re-
search Laboratories, Gaithersburg, Md.). The labeled probe
was purified on a Nensorb column (Nensorb 20; Du Pont
Co., Wilmington, Del.). Labeled probe (5 x 107 cpm) was
added to the hybridization solution (6x SSPE, 1% SDS) and
incubated overnight at 5°C less than the melting temperature
of each probe. After hybridization the membranes were
washed in 6x SSPE-1% SDS three times at room tempera-
ture and once at hybridization temperature. After washing,
the membranes were exposed to Cronex X-ray film (Du
Pont) for 3 to 18 h at -70°C with an intensifying screen.
Dot blot hybridization. Dot blot hybridization was per-
formed as described by Clewley; plasmid pSP321, which
contains the middle one-half (2.7 kilobases) of the B19
genome cloned into the PstI site in the ampicillin resistance
gene of pBR322 (9), was used as a probe. Briefly, 10
sample was added to 200 pul of2x SSC (0.3 M NaCI, 0.03 M
sodium citrate [pH 7.0]), followed by the addition of 200
of 1 M NaCl-0.1 M NaOH. After 10 min at room tempera-
ture, 1.8 ml of 2x SSC was added and then filtered over a
nitrocellulose membrane (Schleicher & Schuell) that had
been prewet with 20x SSC for 30 min. The membrane was
washed briefly in 2x SSC and baked at 80°C for 2 h in a
vacuum oven. The membrane was prehybridized for 4 h at
65°C in 10 ml of hybridization solution (6x SSC, 0.5% SDS,
1x Denhardt solution, 100 ,ug ofsheared salmon sperm DNA
per ml). The membrane was transferred to fresh hybridiza-
tion solution with 106 cpm of [32P]dCT-labeled probe (2 x
106 cpm/,ug). The filter was hybridized overnight at 65°C with
mixing. After hybridization, the filter was washed at 65°C for
2 to 4 h with serial changes oflx SSC-0.1% SDS, allowed to
dry at room temperature for 10 to 20 min, and exposed to
X-ray film. Autoradiograms were developed after 3, 18, and
72 h and 7 days at -70°C with an intensifying screen.
pBS321 was labeled with [32P]dCT by nick translation
(21). Unincorporated [32P]dCT was removed, and the plas-
mid was concentrated with an Centricon 30 filter (W. R.
Grace and Co., Danvers, Mass). Before use, the labeled
plasmid was added to 0.5 ml of sheared salmon sperm DNA
(10 mg/ml), placed in a boiling water bath for 3 min, and then
placed on ice for 5 min.
Serologic assays. IgG and IgM to B19 in human sera were
detected as previously described (4, 14).
Specimens. Serum samples for PCR were obtained from 18
patients at Medical College of Virginia Hospital with hema-
tologic problems compatible with B19 infection. Six ofthe 18
patients had at least onç specimen positive for B19 DNA by
dot blot hybridization, including three patients with sickle
cell anemia and aplastic crisis, two patients with acute
lymphoblastic anemia, and one patient with systemic lupus
erythematosus and autoimmune hemolytic anemia. The
other 12 patients had at least one serum sample containing
IgM to B19; 8 of these 12 had sickle cell disease, 3 had
hereditary spherocytosis, and 1 adult had erthyma infectio-
sum with arthritis.
Additional B19-positive sera were provided by B. J.
Cohen (Wi; London), A. M. Courouce (REM, DES, and
LEC; Paris), and N. Young (Minor; Bethesda, Md.).
Other specimens included ascites and pericardial fluid
from a hydropic fetus and urine and peripheral blood mono-
nuclear cells from a patient with aplastic crisis. Peripheral
blood mononuclear cells were obtained from heparinized
blood samples by using Sepracell-MN (Sepratech Corp.,
Oklahoma City, Okla.); 5 ,ul of the cell suspension was used
in the PCR assay without prior DNA extraction.
For amplification of sera and other body fluids, 1 to 3 ,ul
was added directly to the PCR reaction mixture.
Control serum samples were obtained at random from 20
obstetric patients when blood was drawn for other purposes.
Ail serum was stored at -70°C.
Each of the three primers worked effectively for the
detection of B19 DNA (Tables 1 and 4). Primers Ki and K2
TABLE 2. PCR results of B19 antigen-positive sera tested with
three different sets of primer pairs
Results with primer pair:
J. CLIN. MICROBIOL.
PCR DETECTION OF B19 DNA
FIG. 1. Ethidium bromide-stained agarose gel of PCR amplified
reaction products oftwo different sera (lanes 1 and 2 and lanes 3 and
4). The left lane (unlabeled) contains base pair (bp) marker frag-
ments, with the sizes of two fragments indicated. Undigested (-)
reaction products for both serum samples contained a 699-base-pair
fragment when Ki and K2 were used as primers (lanes 1 and 3) and,
for one ofthe two serum samples, a 442-base-pair fragment when K6
and K7 were used as primers (lane 5). For both serum samples after
PstI digestion (+), two fragments of the predicted length (194 and
505 base pairs) appeared when Ki and K2 were the primers (lanes 2
and 4), but the K6-K7-primed fragment lacked a PstI restriction site
were selected for complete characterization of the PCR
reaction. To find optimal reaction conditions, three anneal-
ing temperatures (37, 40, and 42°C) and three different
numbers of total cycles (25, 30, and 35) were investigated.
The combination of 35 cycles and 37°C produced the best
level of sensitivity and specificity. The expected amplified
product of699 base pairs was observed after amplification of
a dot-blot-positive serum sample (Fig. 1). PstI digestion of
the 699-base-pair fragment generated by primers Ki and K2
in two serum samples yielded a 194-base-pair fragment and a
505-base-pair fragment, as predicted from the nucleotide
sequence (Fig. 1). PCR performed with primers K6 and K7
yielded a DNA fragment of the predicted size, 442 base
pairs, that lacked a PST1 site (Fig. 1). None of the primer
sets produced B19-specific DNA fragments when either
cytomegalovirus DNA, simian virus 40 DNA, or human
cellular DNA (from MRC-5 fibroblasts and from lympho-
cytes) was used.
To determine the sensitivity of PCR, PCR was compared
with dot blot hybridization. The serum from a patient with
aplastic crisis due to human parvovirus B19 was serially
diluted. The dot blot assay detected approximately 0.2 ng of
B19 DNA in 10 ,uI of the diluted serum with an exposure of
the nitrocellulose filter for 7 days at -70°C (Fig. 2). In
contrast, when 1-pul samples of the same dilutions of this
serum were amplified by using PCR and the agarose gels
were stained with ethidium bromide, B19 DNA was detected
in the 1:107 dilution (Fig. 3A). When the agarose gel was
probed with the internal probe K5, both single- and double-
stranded fragments were detected in a 1:1010 dilution of this
serum with an exposure at -70°C for 3 h (Fig. 3B). There-
fore, with ethidium bromide staining the PCR detected
approximately 0.02 pg of B19 DNA, and with a radiolabeled
internal probe the PCR detected 0.02 fg of B19 DNA.
Dot blot hybridization and PCR were compared by using
15 serum samples from six patients with B19 infections. All
FIG. 2. Autoradiogram after dot blot hybridization of plasmid
pSB321 and serum containing B19 virus. Serum and plasmid were
diluted in 2x SSC, and 10 ,ul of each dilution was denatured and
filtered over nitrocellulose. For the serum the fold dilution is
indicated above three of the dots, and for the plasmid the quantity of
DNA on each dot is indicated below three of the dots. 32P-labeled
pSP321 was the probe, and the nitrocellulose sheet was exposed to
X-ray film for 1 week at -70°C.
six patients had at least one serum sample positive for B19
DNA by dot blot hybridization. Of 11 dot-blot-positive
serum samples, all were also positive by PCR. Offour serum
samples negative by dot blot hybridization, three were
positive by PCR. One serum sample was negative by both
tests. In addition we assayed 20 serum samples from 20
pregnant women selected randomly. All 20 serum samples
lacked IgM to B19, and 8 contained IgG to B19. All 20 serum
samples were negative by dot blot hybridization and also
negative by PCR.
Twenty-nine serum samples from 18 patients with proven
B19 infections were assayed for IgM and IgG to B19 and by
PCR (Table 3). Of 22 serum samples that were IgM positive,
17 were PCR positive. All five serum samples (from five
-10 -9 -8
FIG. 3. PCR amplification of serially diluted patient serum.
Samples (1 ,uI) of the same dilutions of the serum used for dot blot
hybridization (Fig. 2) were amplified with primers Ki and K2. After
amplification and agarose gel electrophoresis, the gel was stained
with ethidium bromide (A). The DNA fragments of this gel were
transferred to a nylon membrane and probed with 32P-labeled
internal probe K5 (B). Ethidium bromide stained only the double-
stranded fragment (B) in serum diluted i0-'. The radiolabeled probe
detected single- and double-stranded fragments in serum diluted
10-10 after 3 h of exposure to X-ray film at -70°C.
VOL. 28, 1990
KOCH AND ADLER
TABLE 3. Association of IgM positivity and PCR in sera of
patients infected with B19
No. of serum samples by
PCR result by:
Ethidium bromide staining
patients) that contained IgM but were PCR negative were
obtained during the convalescent phase of illness (.3 days
after the onset of symptoms) and contained IgG to B19.
Seven serum samples from five patients lacked IgM to B19,
and all were PCR positive. One of these five patients was
immunocompromised and had an impaired ability to make
IgG and IgM to B19 (Koch and Adler, in press). The other
four serum samples that lacked IgM but were PCR positive
were from four patients with sickle cell disease and aplastic
crisis. Each ofthese four serum samples was obtained during
acute infection (<4 days after the onset of symptoms) and
lacked IgG to B19.
Of 24 serum samples positive by PCR, 20 were positive
when only ethidium bromide was used to detect B19 DNA
after amplification (Table 3). Four serum samples were
positive only after a radiolabeled probe was used for detec-
tion. These four serum samples (from four patients) were
obtained during the convalescent phase, and each contained
IgG and IgM to B19.
Table 4 compares DNA detection by dot blot hybridiza-
tion and PCR with serologic tests for IgG and IgM to B19 by
duration of illness. Of 10 serum samples obtained 3 days or
less after the onset of illness, 9 were PCR positive but only
6 were IgM reactive. However, all 16 serum samples ob-
tained after 3 days of illness from 16 patients contained IgM
Nine different serum samples with B19 antigen were
subjected to PCR amplification with each ofthe three primer
sets listed in Table 1. After amplification, one serum sample
from France did not react with primers K6 and K7 and one
Richmond isolate did not react with K8 and K9. All the other
isolates reacted with each of the three sets ofprimers (Table
To determine the feasibility of using PCR in a variety of
specimens, partially purified B19 virus was diluted 1:10,000
and added to lymphocytes, urine, and ascitic and pleural
fluids. PCR detected B19 DNA in these specimens. Pleural
TABLE 4. Association between B19 DNA detection and
serologic testing by duration after onset of illness
No. sera positive/no. tested
aIncludes 17 patients. One patient with leukemia who never made antibod-
ies to B19 is excluded.
bAll sera were tested with a radiolabeled probe.
fluid, if used undiluted, inhibited PCR. PCR detected B19
DNA in lymphocyte lysates from a patient with aplastic
crisis due to B19 and in the ascitic and pericardial fluids
obtained from a hydropic fetus.
The PCR with ethidium bromide for detection is at least
104 times more sensitive than dot blot hybridization for the
detection of B19 DNA in serum and detects fewer than 10
genomes with a radiolabeled probe. This level of sensitivity
agrees with a previous report with DNA and RNA probes
(24). Besides sensitivity, PCR has other advantages for
diagnostic laboratories over dot blot hybridization. First, a
radiolabeled probe would be unnecessary for many serum
samples. B19 infections typically produce high titers of virus
in sera, especially during aplastic crisis (6). In this study, 20
serum samples were positive after ethidium bromide stain-
ing, and only 4 serum samples required a radiolabeled probe
for detection of amplified B19 DNA. Second, the procedure
is complete within 8 h. Third, an automated thermocycler for
temperature shifts eliminates personnel time and allows
multiple specimens to be tested simultaneously.
PCR has two disadvantages. First, specimen contamina-
tion occurs easily. Avoiding contamination requires meticu-
lous care in the handling and transfer of specimens. Second,
not all sets of probes will detect all isolates. Of nine isolates
tested against all three sets of probes, two isolates each
failed to react with one set of probes. However, we did find
one set of probes that reacted with all of the isolates. This
was expected, because epidemiologically different isolates of
B19 have variations in restriction enzyme sites, indicating
genetic heterogeneity (B. J. Cohen, personal communica-
PCR is specific and sensitive for detecting B19 viremia but
should be used with IgM detection for the diagnosis of B19
infection, especially when only a single specimen is tested.
Using dot blot hybridization to detect virus, Anderson et al.
showed that B19 viremia precedes the appearance of B19-
specific IgM and that IgM persists for weeks after viremia
has cleared (5). Thus, patient sera obtained early in infection
may lack IgM to B19 but be PCR positive. Later during
infection, sera may be PCR negative but contain IgM. This
agrees with our observations. Four serum samples obtained
early in infection were PCR positive and lacked IgM and
IgG. Five serum samples obtained late in infection were IgM
and IgG positive and PCR negative.
Finally, we and others have recently observed immuno-
compromised patients with chronic B19 infections (15, 16;
Koch and Adler, in press). These chronic infections occur
because ofthe inability ofthese patients to produce adequate
levels of IgG or IgM antibodies to B19; antigen or DNA
detection is required for diagnosis. For these immunocom-
promised patients PCR, because of its excellent sensitivity
and specificity, is likely to be the most useful ofthe currently
available diagnostic tests.
This work was supported by a grant from the A.D. Williams
Foundation, Medical College of Virginia.
We thank N. Young, B. Cohen, J. P. Clewley, A. Courouce, and
L. J. Anderson for sera and plasmids and G. Buck and T. Reynolds,
Medical College of Virginia-Virginia Commonwealth University
Nucleic Acid Core Laboratory, for assistance with DNA synthesis.
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VOL. 28, 1990