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Peptide nucleic acid-based in situ hybridization assay for detection of parvovirus B19 nucleic acids.

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, single-base 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 (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 [10.sup.7] 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 [10.sup.4] viral genome-equivalents/ cell. After adsorption for 2 h at 37[degrees]C, we removed the inoculum virus by washing and incubated the cells at 37[degrees]C and 5% [CO.sub.2] 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 samples, 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 PCR-ELISA.

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 phosphate-buffered saline (PBS 137 mmol/L NaCl, 10 mmol/L [Na.sub.2]HP[O.sub.4], 2 mmol/L K[H.sub.2]P[O.sub.4], 2.7 mmol/L KCI). Bone marrow-derived mononuclear cells were isolated by centrifugation on Ficoll-Pague 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[degrees]C for at least 4 weeks or at -20[degrees]C for at least 1 year.

Fixed cells and dewaxed sections were digested at 37[degrees]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; Gen-Bank accession no. NC 000883).

BIOTINYLATED PARVOVIRUS B19 PNA PROBE

The PNA probe used ended with 8-amino-3,6-dioxaoctanoic acid (-OO-) and was labeled at the N[H.sub.] 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).

DIGOXIGENIN-LABELED PARVOVIRUS B19 DNA PROBE

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 [micro]L final volume) containing 50 mM KCI, 2.5 mM MgC[l.sub.2], 10 mM Tris-HCI (pH 8.3), 10x 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[degrees]C for 5 min, followed by 40 cycles of denaturation at 95[degrees]C for 30 s, annealing at 52[degrees]C for 30 s, extension at 72[degrees]C for 1 min, and a final extension step at 72[degrees]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 [micro]L of hybridization solution containing the PNA biotinylated probe at different concentrations (10, 20, 50, and 100 pmol/ reaction), prewarmed at 50[degrees]C for 10 min to avoid self-aggregation. Two hybridization mixtures were tested: a home-made solution consisting of 500 nL/[micro]L deionized formamide, 100 ng/[micro]L dextran sulfate, and 250 ng/[micro]L carrier calf thymus DNA in 2x 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[degrees]C for 5 min and then incubated at 55[degrees]C for different hybridization times (0.5, 1, 3, and 12 h). After hybridization, specimens were washed at 55[degrees]C for 25 min in 1 x 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-HCI (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-HCI (pH 7.3) containing 150 mmol/L NaCl and finally equilibrated in 100 mmol/L Tris-HCI (pH 9.5) containing 100 mmol/L NaCl and 50 mmol/L MgC[l.sub.2]. The alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl phosphate /nitrotetrazolium 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.

DNA-ISH ASSAY

Hybridization reactions were carried out in 25 [micro]L of a home-made hybridization solution (500 nL/[micro]L deionized formamide, 100 ng/[micro]L dextran sulfate, and 250 ng/[micro]L carrier calf thymus DNA in 2x SSC buffer) containing 20 ng of digoxigenin-labeled B19 DNA probe. Specimens and the hybridization mixture were denatured together by heating at 95[degrees]C for 5 min and then incubated at 37[degrees]C for 12 h. After hybridization, specimens were washed twice at 37[degrees]C with 500 mL/L formamide and 2x SSC buffer and twice at room temperature in 2x 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 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.

[FIGURE 1 OMITTED]

The biotinylated PNA probe and the digoxigenin-labeled DNA probe detected B19 genome in 1319-infected UT-7/EpoS1 cells (Fig. 1, A and C). Moreover, mock-infected 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 1319-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 1319-infected and mock-infected UT-7/EpoS1 cells.

[FIGURE 2 OMITTED]

Having established the performance of the B19 PNA-ISH 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, 1319-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 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. 213; 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 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.

This work was supported by funds from Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR) and from the University of Bologna.

Received December 2, 2005; accepted March 9, 2006.

Previously published online at DOI: 10.1373/clinchem.2005.064741

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[1] Nonstandard abbreviations: PNA, peptide nucleic acid; ISH, in situ hybridization; PBS, phosphate-buffered saline; and SSC, standard saline citrate.

Francesca Bonvicini, Claudia Filippone, Elisabetta Manaresi, Giovanna Angela Gentilomi, Marialuisa Zerbini, Monica Musiani, And Giorgio Gallinella *

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.gall inella@unibo.it.
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Title Annotation:Molecular Diagnostics and Genetics
Author:Bonvicini, Francesca; Filippone, Claudia; Manaresi, Elisabetta; Gentilomi, Giovanna Angela; Zerbini,
Publication:Clinical Chemistry
Date:Jun 1, 2006
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