Clinical Chemistry 52:6
973–978 (2006)
Molecular Diagnostics
and Genetics
Peptide Nucleic Acid–Based In Situ Hybridization
Assay for Detection of Parvovirus B19
Nucleic Acids
Francesca Bonvicini, Claudia Filippone, Elisabetta Manaresi,
Giovanna Angela Gentilomi, Marialuisa Zerbini, Monica Musiani, and
Giorgio Gallinella*
Background: Peptide nucleic acid (PNA) molecules are
known to bind complementary nucleic acid sequences
with a much stronger affinity and with more stable
binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic
acids by in situ hybridization assay (ISH) and/or PCR, as
an experimental model to develop an ISH assay that
uses biotinylated PNA probes to detect viral genome in
clinical specimens.
Methods: We first optimized the PNA-ISH assay on
B19-infected and mock-infected UT-7/EpoS1 cells and
then tested the assay on archival B19 specimens and on
consecutive specimens. All data were compared with
data obtained with a standardized DNA-based ISH
assay and confirmed by a PCR-ELISA.
Results: PNA-ISH detected B19 genome in a higher
number of B19-infected UT-7/EpoS1 cells and with a
more defined localization of viral nucleic acids than the
standardized DNA-ISH assay. Moreover, PNA-ISH was
able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH
detected more positive samples than DNA-ISH when
consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results.
Conclusions: The PNA-ISH assay had sensitivity and
specificity comparable to a PCR assay and was more
practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic
test for the detection of viral nucleic acids in clinical
specimens.
© 2006 American Association for Clinical Chemistry
In situ hybridization (ISH)1 techniques are powerful tools
for cytogenetic analysis. Many labeled DNA or RNA
probes and hybridization protocols are available to detect
target nucleic acids of pathogens, especially viruses in
clinical specimens, but usually these techniques are laborious.
The need for more practical and sensitive detection of
target viral nucleic acids in infected cells prompted us to
explore the hybridization characteristics of probes consisting of peptide nucleic acid (PNA), a synthetic molecule
that structurally imitates DNA (1 ). In PNA molecules, the
negatively charged sugar-phosphate backbone of DNA is
replaced by an achiral, neutral polyamide backbone
formed by repetitive units of N-(2-aminoethyl)glycine.
The uncharged nature of PNA is responsible for the
enhanced thermal stability of PNA–DNA and PNA–RNA
duplexes compared with the corresponding DNA–DNA,
DNA–RNA, and RNA–RNA hybrids. As a result, singlebase mismatches have a considerably more destabilizing
effect, and short PNA probes ensure high specificity (2 ).
Unlike DNA, PNA is stable across wide ranges of temperature and pH and is resistant to nucleases and proteases (3 ). Because of its biostability, PNA was first used
in genetic procedures as an antigene and antisense agent
to inhibit both eukaryotic translation and transcription of
target genes (4, 5 ). Many chemistry, biology, and biotechnology research areas have obtained extraordinary results
by use of PNA in a wide variety of hybridization formats
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Bologna, Italy.
* Address correspondence to this author at: Department of Clinical and
Experimental Medicine, Division of Microbiology, University of Bologna,
Via Massarenti 9, 40138 Bologna, Italy. Fax 39-51-307397; e-mail giorgio.
[email protected].
Received December 2, 2005; accepted March 9, 2006.
Previously published online at DOI: 10.1373/clinchem.2005.064741
1
Nonstandard abbreviations: PNA, peptide nucleic acid; ISH, in situ
hybridization; PBS, phosphate-buffered saline; and SSC, standard saline citrate.
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Bonvicini et al.: PNA-ISH Assay for Parvovirus B19
(6, 7 ) and as a detector in PCR and real-time PCR methods
(8 ).
In recent years, PNA technology has focused on the
improvement of PNA as a diagnostic tool in chromosomal
analysis and microbiology for the identification and characterization of prokaryotes (9 –13 ) and in virology for the
detection of Epstein–Barr-encoded RNAs (14 ).
The model system of human parvovirus B19 (B19) has
been studied to investigate the use of PNA molecule as
a probe in ISH assays for the detection of viral nucleic
acids in clinical specimens. B19 of the family Parvoviridae
is the etiologic agent responsible for a wide range of
clinical syndromes, such as erythema infectiosum, postinfection arthropathies, fetal hydrops, transient aplastic
crises in patients with hemolytic anemia, and chronic
infections, mainly in immunocompromised patients. Because B19 cannot grow efficiently in established cell
cultures, its diagnosis relies mainly on the detection of
nucleic acids in clinical specimens by ISH assay, if localization of the virus is required, or by PCR.
We developed a new, simplified, and robust protocol
for a PNA-based ISH assay for the detection of B19
genome in clinical specimens and compared it with a
standardized DNA-based ISH assay.
Materials and Methods
UT-7/EpoS1 cells and B19 virus infection
To develop an ISH assay for B19 nucleic acids using a
PNA probe, we prepared B19-infected and mock-infected
UT-7/EpoS1 cells (15 ). For infection, UT-7/EpoS1 cells
were incubated at density of 107 cells/mL in RPMI 1640 in
the presence of a B19 viremic serum, previously identified
in our laboratory, to obtain a multiplicity of infection of
104 viral genome-equivalents/cell. After adsorption for
2 h at 37 °C, we removed the inoculum virus by washing
and incubated the cells at 37 °C and 5% CO2 in RPMI 1640
containing 100 mL/L fetal calf serum and 2 kIU/L erythropoietin, at an initial density of 106 cells/mL, and then
harvested the cells at 48 h post infection. Under these
conditions, ⬃10%–20% of cells can be productively infected by B19 virus as determined by indirect immunofluorescence assay using monoclonal antibodies directed
against viral capsid proteins.
archival and clinical samples
Positive and negative archival samples were collected in
our laboratory in the year 2004 and tested previously by
PCR-ELISA for B19 virus DNA (16 ). The positive archival
samples comprised 5 bone marrow aspirates from anemic
patients whose blood previously tested positive for B19
DNA by PCR-ELISA, 5 amniotic fluid cell samples from
patients with fetal hydrops whose amniotic fluid previously tested positive for B19 DNA by PCR-ELISA, and 3
paraffin-embedded liver biopsy sections from transplant
recipients whose biopsy lysates previously tested positive
for B19 DNA by PCR-ELISA. As negative archival sam-
ples, we analyzed 3 bone marrow aspirates, 2 amniotic
fluid cell samples, and 2 paraffin-embedded liver sections
from patients with pathologies unrelated to B19, which
had previously tested negative for B19 DNA by PCRELISA.
We also prospectively analyzed 15 consecutive clinical cellular specimens (10 bone marrow aspirates and 5
amniotic fluid cell samples) sent to our laboratory during
a B19 epidemic period (March to June 2005) with a clinical
suspicion of B19 virus infection.
The PCR-ELISA used as the comparison method is a
competitive PCR based on coamplification of the viral
target and of a mutagenized competitor target that acts as
internal control. The amplification products are labeled by
incorporation of digoxigenin and detected by hybridization with biotinylated DNA oligonucleotide probes, followed by capture on streptavidin-coated microtiter plate
wells and immunoenzymatic detection of the digoxigenin
moiety. The method is absolutely specific for B19 virus
and is able to detect 10 –100 genome copies/reaction.
specimen preparation and proteolytic
treatments
B19-virus–infected and mock-infected UT-7/EpoS1
cells were harvested and washed 3 times in phosphatebuffered saline (PBS 137 mmol/L NaCl, 10 mmol/L
Na2HPO4, 2 mmol/L KH2PO4, 2.7 mmol/L KCl). Bone
marrow– derived mononuclear cells were isolated by centrifugation on Ficoll-Paque PLUS (Pharmacia), and cells
from amniotic fluid specimens were harvested by centrifugation and washed in PBS buffer.
Cells were then smeared on silanated glass slides and
fixed with 40 g/L paraformaldeyde in PBS for 10 min.
After fixation, samples were washed 3 times in PBS (5 min
each), dehydrated with ethanol washes (300, 600, and 950
mL/L; 2 min each) and then air dried (17 ).
Paraffin-embedded tissue sections were dewaxed sequentially in 2 changes of fresh xylene (10 min each),
washed in absolute ethanol for 5 min, and then air dried.
Cells and sections could be stored at 4 °C for at least 4
weeks or at ⫺20 °C for at least 1 year.
Fixed cells and dewaxed sections were digested at
37 °C with a prewarmed solution of 1 mg/L pepsin in
0.01 mol/L HCl for 10 min and 250 mg/L pepsin in
0.1 mol/L HCl for 30 min, respectively. After digestion,
samples were washed 3 times in PBS (5 min each), and
then dehydrated with ethanol washes (300, 600, and 950
mL/L; 2 min each).
biotinylated parvovirus b19 dna probe
A 20-bp 5⬘-biotinylated B19 DNA probe (5⬘-biotin-ATGCAGCTACAACTTCGGAG-3⬘) was used. The probe sequence was designed to be complementary to a central
region of the B19 genome (nucleotides 2294 –2313; GenBank accession no. NC_000883).
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Clinical Chemistry 52, No. 6, 2006
biotinylated parvovirus b19 pna probe
The PNA probe used ended with 8-amino-3,6-dioxaoctanoic acid (-OO-) and was labeled at the NH2 terminus with biotin (Applied Biosystems). The 16-oligomer
PNA probe sequence (5⬘-biotin-OO-GCAGCTACAACTTCGG-3⬘) was designed to be complementary to a central
region of the B19 genome (nucleotides 2296 –2311).
tetrazolium blue chloride; Roche) was then added according to the manufacturer’s instructions. The reaction was
allowed to proceed for 15–30 min, and the development of
a dark blue precipitate at the enzyme site in positive cells
was monitored by microscopic examination.
This same protocol was also used for hybridization
using the 20-oligomer biotinylated DNA probe.
digoxigenin-labeled parvovirus b19 dna probe
dna-ish assay
The digoxigenin-labeled B19 DNA probe was prepared by
incorporating digoxigenin-labeled dUTP in a PCR reaction. Briefly, the amplification reaction was carried out as
follows: 5 ng of B19 DNA target (plasmid containing B19
genome) was added to a reaction mixture (50 ␮L final
volume) containing 50 mM KCl, 2.5 mM MgCl2, 10 mM
Tris-HCl (pH 8.3), 10⫻ DIG DNA labeling Mix (Roche), 2
U of FastStart Taq DNA polymerase (Roche), and 0.1 M
each of K8 (nucleotides 4882– 4900; 5⬘-AGCTACAGATGCAAAACAA-3⬘) and K9 (nucleotides 5190 –5172; 5⬘TAACCACAACAAATGTTTA-3⬘) primers, which recognize a sequence of B19 structural proteins (18 ). The
cycling profile consisted of an initial denaturation step
at 95 °C for 5 min, followed by 40 cycles of denaturation
at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at
72 °C for 1 min, and a final extension step at 72 °C for
5 min. The amplified 308-bp product was checked by
electrophoresis and then purified from the PCR mixture
by use of the QIAquick PCR Purification Kit (Qiagen),
according to the manufacturer’s instructions.
pna-ish assay
The hybridization reaction was carried out in 25 ␮L of
hybridization solution containing the PNA biotinylated
probe at different concentrations (10, 20, 50, and 100 pmol/
reaction), prewarmed at 50 °C for 10 min to avoid selfaggregation. Two hybridization mixtures were tested: a
home-made solution consisting of 500 nL/␮L deionized
formamide, 100 ng/␮L dextran sulfate, and 250 ng/␮L
carrier calf thymus DNA in 2⫻ standard saline citrate
(SSC) buffer (300 mmol/L NaCl, 30 mmol/L sodium
citrate, pH 7.0) (17 ), and a commercially available hybridization solution (Dako). Specimens and the hybridization
mixture were denatured together by heating at 95 °C for
5 min and then incubated at 55 °C for different hybridization times (0.5, 1, 3, and 12 h). After hybridization,
specimens were washed at 55 °C for 25 min in 1⫻ Stringent Wash (Dako).
For the detection of hybridized probes, specimens were
incubated for 30 min with streptavidin conjugated to
alkaline phosphatase (Roche), diluted 1:500 in 100 mmol/L
Tris-HCl (pH 7.3) containing 150 mmol/L NaCl and 10 g/L
Blocking Reagent (Roche). After incubation, specimens
were washed twice with 100 mmol/L Tris-HCl (pH 7.3)
containing 150 mmol/L NaCl and finally equilibrated in
100 mmol/L Tris-HCl (pH 9.5) containing 100 mmol/L
NaCl and 50 mmol/L MgCl2. The alkaline phosphatase
substrate (5-bromo-4-chloro-3-indolyl phosphate/nitro-
Hybridization reactions were carried out in 25 ␮L of a
home-made hybridization solution (500 nL/␮L deionized
formamide, 100 ng/␮L dextran sulfate, and 250 ng/␮L
carrier calf thymus DNA in 2⫻ SSC buffer) containing
20 ng of digoxigenin-labeled B19 DNA probe. Specimens
and the hybridization mixture were denatured together
by heating at 95 °C for 5 min and then incubated at 37 °C
for 12 h. After hybridization, specimens were washed
twice at 37 °C with 500 mL/L formamide and 2⫻ SSC
buffer and twice at room temperature in 2⫻ SSC buffer
(10 min each). Colorimetric detection of hybridized
probes was performed as described previously, using
sheep polyclonal anti-digoxigenin Fab fragments conjugated to alkaline phosphatase (Roche) instead of streptavidin.
evaluation of results
Results of the ISH assays are reported as either positive or
negative based on the presence and distribution of the
staining. All clinical samples were analyzed in duplicate
in 2 different runs performed on different days and
evaluated in a blinded approach by different operators.
Results
As a first step in confirming that the PNA-ISH assay did
indeed detect B19 nucleic acids by use of a biotinylated
PNA probe, we tested B19-infected and mock-infected
UT-7/EpoS1 cells.
We tested different concentrations of PNA probe (from
10 to 100 pmol/reaction), hybridization times (from 30 min
to 12 h), and 2 hybridization mixtures (the commercially
available solution from Dako and the home-made mixture) on B19-infected UT-7/EpoS1 cells. On the basis of
visual analysis, 10 pmol of biotinylated PNA probe for
reaction and 1 h of hybridization time were the optimal
conditions for the PNA-ISH assay, providing clear localization of B19 nucleic acids in infected UT-7/EpoS1 cells
(Fig. 1A). Hybridization for more than 1 h did not lead to
an increase in B19 genome detection. Moreover, we found
no differences in hybridization signal with either hybridization solution. Mock-infected UT-7/EpoS1 cells were
completely negative at all probe concentrations and reaction times tested and with both hybridization solutions
(Fig. 1B).
Having optimized the PNA-ISH conditions, we next
compared the performance of the short biotinylated PNA
probe with that of a long (308-bp) digoxigenin-labeled
DNA probe (used as the comparison probe) and the
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Bonvicini et al.: PNA-ISH Assay for Parvovirus B19
Fig. 1. PNA-ISH assay of B19-infected (A)
and mock-infected UT-7/EpoS1 cells (B);
and DNA-ISH assay of infected B19-infected
UT-7/EpoS1, using digoxigenin-labeled DNA
probe (C) and biotinylated DNA probe (D).
performance of a short biotinylated DNA probe (20 bp)
that had the same oligonucleotide sequence as the PNA
probe. The 16-oligomer biotinylated PNA probe and the
20-oligomer biotinylated DNA probe were used according to the PNA-ISH protocol, whereas the long digoxigenin-labeled DNA probe was used in the standardized
DNA-ISH assay.
The biotinylated PNA probe and the digoxigeninlabeled DNA probe detected B19 genome in B19-infected
UT-7/EpoS1 cells (Fig. 1, A and C). Moreover, mockinfected UT-7/EpoS1 cells were completely negative
with both probes. When we performed a quantitative
comparison between the PNA-ISH and DNA-ISH assays,
using the same batch of B19-infected UT-7/EpoS1 cells in
5 different experiments, a mean of 21 of 100 counted cells
were positive by the biotinylated PNA probe vs a mean of
10 of 100 cells with the digoxigenin DNA probe.
The 20-oligomer biotinylated DNA probe did not distinguish positive from negative cells (Fig. 1D): a faint,
nonspecific signal was detected in both B19-infected and
mock-infected UT-7/EpoS1 cells.
Fig. 2. PNA-ISH assay of B19-positive archival
bone marrow cells (A) and a B19-positive
archival liver biopsy (Methyl Green counterstain; B).
Having established the performance of the B19 PNAISH assay on cellular specimens, we analyzed archival
specimens (5 bone marrow aspirates, 5 amniotic fluids,
and 3 liver tissue sections), collected in our laboratory in
2004 and previously testing positive for B19 DNA by
PCR-ELISA, with the PNA-ISH and the standardized
DNA-ISH assays in duplicate in 2 different runs. In each
run, B19-infected UT-7/EpoS1 and mock-infected UT-7/
EpoS1 cells were tested as positive and negative controls,
respectively.
The 5 bone marrow aspirates that had previously tested
positive for B19 DNA by PCR-ELISA were positive at both
assays (Fig. 2A; also see Table 1 in the Data Supplement that
accompanies the online version of this article at http://
www.clinchem.org/content/vol52/issue6/).
Of the 5 amniotic fluid cell samples that previously
tested positive for B19 DNA by PCR-ELISA from patients
with a clinical diagnosis of B19 fetal hydrops, all were
positive in the B19 PNA-ISH assay, whereas only 2
samples were positive in the DNA-ISH assay (see Table 1
in the online Data Supplement). The results obtained in
Clinical Chemistry 52, No. 6, 2006
the 2 runs performed on different days were completely
concordant.
Among 3 archival liver biopsies that had previously
tested positive for B19 DNA by PCR-ELISA of the biopsy
lysates, 3 were positive in the PNA-ISH assay, whereas
only 1 was positive in the DNA-ISH assay (Fig. 2B; also
see Table 1 in the online Data Supplement). The results
obtained in the 2 runs performed on different days were
completely concordant, and the absence of endogenous
liver biotin was assessed by hybridization assays omitting
the biotinylated probe.
Negative archival samples (3 bone marrow aspirates,
2 amniotic fluid cell samples, and 2 liver biopsies) were
completely and consistently unstained in both assays.
To investigate the suitability of the new PNA-ISH as a
diagnostic tool, we analyzed 15 consecutive clinical specimens sent to our laboratory during a B19 epidemic
(March to June 2005) to detect B19 genome with both the
PNA-ISH and DNA-ISH assays performed in the same
run. The assays were performed in duplicate and evaluated in a blinded approach by different operators.
Ten consecutive bone marrow aspirates were analyzed:
6 were positive and 4 were negative in the PNA-ISH
assay, whereas 3 were positive and 7 were negative in
DNA-ISH assay (see Table 2 in the online Data Supplement).
When we analyzed 5 amniotic fluid cell specimens, 2
were positive and 3 were negative for B19 DNA in both
the PNA-ISH and DNA-ISH assays (see Table 2 in the
online Data Supplement), but the PNA probe was able to
identify a higher number of positive cells in positive
clinical samples than the DNA probe.
The 15 consecutive clinical specimens were analyzed at
the same time with the PCR-ELISA to detect B19 DNA
(see Table 2 in the online Data Supplement). PCR-ELISA
results were completely in agreement with the PNA-ISH
results: 6 bone marrow aspirates were positive and 4 were
negative, whereas 2 amniotic fluid specimens were positive and 3 were negative.
Discussion
ISH assays for the diagnosis of B19 virus infection can
yield information on both the presence and distribution of
viral nucleic acids with preservation of cellular and tissue
morphology; they therefore can integrate the results obtained from nucleic acid amplification techniques. Our
ISH assay, which uses a biotinylated PNA probe suitable
for the detection of B19 genome in a routine diagnostic
laboratory, enabled colorimetric detection of target with a
light microscope, thus eliminating the need for a fluorescence microscope.
ISH with the PNA probe detected a higher number of
positive cells and a more defined localization of viral
nucleic acids than the standardized DNA probes. This
result can be explained by the greater permeability of the
short PNA probe through cytoplasmic and nuclear membranes with respect to the long probe. Moreover, unlike
977
cloned or PCR-labeled probes, the single-stranded PNA
probe eliminates the possibility of annealing with complementary strands in the hybridization solution. When
the short, single-stranded biotinylated DNA probe was
tested, no specific signal was detected in spite of the fact
that the sequence of the DNA probe was the same as that
of the PNA probe. It is evident that the short DNA probe
is unable to ensure specific binding under our hybridization conditions. Unlike the PNA probe, which was stable
across wide pH and temperature ranges, the DNA probe
required the use of stringent conditions, making the
hybridization reaction precarious.
To investigate the suitability of the PNA probe, we first
performed the novel PNA-ISH assay and the standardized DNA-ISH assay with archival specimens (cell samples and tissue sections) that previously tested positive for
B19 DNA by PCR-ELISA and then with 15 consecutive
clinical specimens sent to our laboratory with a clinical
suspicion of B19 virus infection.
When positive archival specimens (5 bone marrow
aspirates, 5 amniotic fluid cell samples, and 3 liver biopsies) were analyzed with the 2 ISH assays, detection of
B19 genome on bone marrow aspirates was completely
concordant, whereas for amniotic fluid cell samples and
tissue sections, the results were discordant. In particular,
the PNA probe detected B19 genome in all positive
archival samples, whereas the DNA probe failed to detect
B19 nucleic acids in 3 amniotic fluid cell samples and 2
tissue sections. These results suggest that the PNA molecule might be a better probe for detecting B19 genome in
archival specimens and in tissue specimens: the short
length of the PNA probe allows the hybridization of even
fragmentized copies of B19 genome in clinical specimens
that experienced target degradation and degeneration
during fixation and cutting procedures.
When we analyzed 15 consecutive clinical specimens,
the PNA-ISH assay detected B19 viral genome in 8 of the
specimens, whereas the DNA-ISH assay detected B19
viral genome in 5 specimens. We also compared the
results obtained with our assay with the results obtained
by PCR-ELISA and found close agreement.
The novel PNA-ISH assay is simple and quick to
perform, and all reagents used in the assay are commercially available. Thus, it may be a practical and reliable
test for the diagnosis of B19 infection in clinical specimens.
At present, the cost of PNA synthesis represents an
important initial outlay for laboratories, but analysis of
the cost/benefit ratio showed that cost of the PNA-ISH
assay is competitive with that of the DNA-ISH assay: no
additional materials or costly instruments are required to
prepare probes and to detect hybrids. Moreover, the novel
PNA-ISH assay provides saves time and enhances the
sensitivity of B19 genome detection with respect to traditional hybridization assays.
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Bonvicini et al.: PNA-ISH Assay for Parvovirus B19
This work was supported by funds from Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR) and
from the University of Bologna.
References
1. Nielsen PE, Egholm M, Berg RH, Buchardt O. Sequence-selective
recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 1991;254:1497–500.
2. Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM,
Driver DA, et al. PNA hybridizes to complementary nucleotides
obeying the Watson-Crick hydrogen-bonding rules. Nature 1993;
365:566 – 8.
3. Demidov VV, Potaman VN, Frank-Kameneskii MD, Egholm M,
Buchard O, Sonnichsen SH, et al. Stability of peptide nucleic acids
in human serum and cellular extracts. Biochem Pharmacol 1994;
48:1310 –3.
4. Hanvey JC, Peffer NJ, Bisi JE, Thomson SA, Cadilla R, Josey JA, et
al. Antisense and antigene properties of peptide nucleic acids.
Science 1992;258:1481–5.
5. Nielsen PE, Egholm M, Berg RH, Buchardt O. Peptide nucleic acids
(PNAs): potential antisense and anti-gene agents. Anticancer Drug
Des 1993;8:53– 63.
6. Perry-O’Keefe H, Yao XW, Coull JM, Funchs M, Egholm M. Peptide
nucleic acid pre-gel hybridization: an alternative to southern
hybridization. Proc Natl Acad Sci U S A 1996;93:14670 –5.
7. Nielsen PE. PNA technology. Mol Biotechnol 2004;26:233– 48.
8. Svanvik N, Stahlberg A, Sehlstedt U, Sjoback R, Kubista M.
Detection of PCR-ELISA products in real time using light-up probes.
Anal Biochem 2000;287179 – 82.
9. Stender H, Lund K, Petersen KH, Rasmussen OF, Hongmanee P,
Miorner H, et al. Fluorescent in situ hybridization assay using
peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of
mycobacterium cultures. J Clin Microbiol 1999;37:2760 –5.
10. Lehtola MJ, Loades CJ, Keevil CW. Advantages of peptide nucleic
acid for sensitive site directed 16S rRNA fluorescent in situ
hybridization (FISH) detection of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari. J Microbiol Methods 2005;
62:211–9.
11. Wilson DA, Joyce MJ, Hall LS, Reller LB, Roberts GD, Hall GS, et
al. Multicenter evaluation of a Candida albicans peptide nucleic
acid fluorescent in situ hybridization probe for characterization of
yeast isolates from blood cultures. J Clin Microbiol 2005;43:
2909 –12.
12. Perry-O’Keefe H, Rigby S, Oliveira K, Sorensen D, Stender H, Coull
J, et al. Identification of indicator microorganisms using a standardized PNA FISH method. J Microbiol Methods 2001;47:281–
92.
13. Oliveira K, Procop GW, Wilson D, Coull J, Stender H. Rapid
identification of Staphylococcus aureus directly from blood cultures by fluorescence in situ hybridization with peptide nucleic
acid probes. J Clin Microbiol 2002;40:247–51.
14. Engel PJ. Absence of latent Epstein-Barr virus in thymic epithelial
tumors as demonstrated by Epstein-Barr-encoded RNA (EBER) in
situ hybridization. APMIS 2000;108:393–7.
15. Shimomura S, Komatsu N, Frickhofen N, Anderson S, Kajigaya S,
Young NS. First continuous propagation of B19 parvovirus in a cell
line. Blood 1992;79:18 –24.
16. Bonvicini F, Gallinella G, Cricca M, Venturoli S, Musiani M, Zerbini
M. A new primer set improves the efficiency of competitive
PCR-ELISA for the detection of B19 DNA. J Clin Virol 2004;30:
134 – 6.
17. Gentilomi GA, Zerbini M, Musiani M, Gallinella G, Gibellini D,
Venturoli S, et al. In situ detection of B19 DNA in bone marrow of
immunodeficient patients using a digoxigenin-labeled probe. Mol
Cell Probes 1993;7:19 –24.
18. Kock WC, Adler SP. Detection of human parvovirus B19 DNA by
using the polymerase chain reaction. J Clin Microbiol 1990;28:
65–9.