INTRODUCTION

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An important first step in the molecular detection of hepatitis C virus (HCV) is the extraction and purification of RNA from clinical samples. Conventional sample preparation methods usually involve phenol-chloroform extraction and precipitation steps (3) which are not ideal in the development of automated HCV detection systems. Magnetic beads offer one alternative and have been used in efforts to develop closed diagnostic systems. Such magnetic particles generally allow for a rapid change of reaction buffers and reagents simply by applying a magnetic field, thereby circumventing centrifugation or precipitation steps (6, 12, 20, 26). For example, in a previous study a hybridization capture assay for HCV based on magnetic beads was developed (23). However, although the protocol was rapid, it showed a lower sensitivity than the conventional extraction procedure, with the capture efficiency varying considerably depending on the particular probe used. These HCV capture probes were designed to hybridize to the 5' nontranslated region (NTR), which is a highly conserved region but which is also predicted to have a high degree of secondary structure that may affect the efficiency of hybridization (2, 4, 9).

In a recent study, using analytical biosensor technology, we investigated whether nucleic acid capture could be improved by taking advantage of "stacking hybridization" (16). This refers to the stabilizing effect that exists between DNA oligonucleotides when they hybridize in a contiguous tandem fashion to single stranded complementary DNA (8, 11). We designed adjacently positioned oligonucleotide probes, one of which was prehybridized in solution to a target while the other was immobilized on a chip surface in the biosensor (16). It was found that when such probes anneal adjacently on the HCV template they interact cooperatively, increasing the capture efficiency probably through a base stacking effect and/or the suppression of secondary structure. The capture efficiency was also notably decreased when gaps between the probes were introduced.

Here, we describe an HCV capture PCR assay based on the use of such cooperatively interacting oligonucleotide probes. This sample preparation method uses magnetic beads as a solid support, allowing the method to be easily adapted for automatic pipetting work stations. The applicability of this assay was investigated first by performing a comparative study with a commercial quantitative assay and second by quantifying the RNA extracted by this capture method and a conventional extraction procedure.

	MATERIALS AND METHODS

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Cooperative oligonucleotides in capture of HCV rDNA. (i) Preparation of HCV rDNA. HCV RNA (genotypes 1a, 2b, and 3a) was extracted from serum samples from infected individuals, and reverse transcription (RT)-PCR was carried out as described previously (26). The resulting PCR product containing the 5' NTR of HCV (nucleotides [nt] 18 to 341 [4]) was subcloned into the pGEM-T vector (Promega, Madison, Wis.) and then transferred into the polylinker of plasmid pGEM-4Z (Promega). The identity of the final construct was confirmed by DNA sequence analysis. Single-stranded recombinant DNA (rDNA) targets were prepared by PCR amplification of the 5' NTR of HCV cloned in pGEM-4Z with the HCV-specific primers OU49 (5'-GGCGACACTCCACCATGAATC-3' [nt 18 to 38]) and OD66 (5'-biotin-GGTGCACGGTCTACGAGACC-3' [nt 322 to 341]), and the resulting biotinylated 324-bp PCR fragment was immobilized onto streptavidin-coated paramagnetic beads (Dynabeads M-280 Streptavidin; Dynal AS, Oslo, Norway). By strand-specific elution of the nonbiotinylated strand (6), a pure single-stranded template for hybridization was obtained, as described previously (16). The single-stranded HCV rDNA target was then fivefold terminally titrated in a buffer containing 0.2 µg of yeast RNA (Boehringer, Mannheim, Germany) per µl and stored at 20°C.

(ii) Magnetic-bead capture and detection of HCV rDNA by PCR. Super paramagnetic beads (10 mg/ml) covalently coupled with an 18-mer HCV-specific capturing probe, C1 (5'-GGTGCACGGTCTACGAGA-3' [nt 324 to 341]), were conditioned by two washes in binding-washing (B/W) buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 M NaCl, 1 mM -mercaptoethanol, 0.1% Tween 20). To reduce nonspecific adsorption of nucleic acids, 1 µg of yeast RNA was added to the beads, which were then resuspended in 6× SSPE (0.9 M NaCl [pH 7.4], 60 mM NaH₂PO₄, 7.5 mM EDTA) to a final concentration of 10 mg/ml. A prehybridization procedure was performed by incubation of 30 µl of single-stranded rDNA at 54°C for 15 min in 100 µl of 6× SSPE containing 1 µg of yeast RNA and 0.5 µM 18-mer probe P1 (5'-CCTCCCGGGGCACTCGCA-3' [nt 306 to 323]). Control samples without this prehybridizing probe were prepared in parallel. The DNA samples were then incubated with 250 µg of the previously prepared magnetic beads (coupled with the HCV-specific capture probe C1) for 90 min at room temperature with constant rotation. After the hybridization step, the beads were washed three times in 100 µl of B/W buffer, twice in 100 µl of 10× PCR buffer (Perkin-Elmer, Foster City, Calif.) (and changed to a new microcentrifuge tube prior to the final washing step), and resuspended in 100 µl of H₂O. A control sample without added target rDNA (which was incubated with the beads and underwent the washing steps, etc.) was included to monitor cross-contamination. Five microliters of resuspended beads was used as template in a PCR amplification performed with 0.2 µM OU49 and the prehybridizing probe (P1) in a 50-µl reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl₂, 0.2 mM (each) deoxynucleoside triphosphates and 0.5 U of AmpliTaq DNA polymerase (Perkin-Elmer). The mixture was overlaid with 50 µl of light mineral oil (Sigma Chemical Co., St. Louis, Mo.). PCR was performed with a Perkin-Elmer 9600 thermocycler using a temperature profile of 94°C for 1.5 min, followed by 35 cycles of 94°C for 25 s, 62°C for 30 s, and 72°C for 1 min, and ending with 72°C for 10 min. To permit a quantitative comparison of the end-diluted series of rDNA, seminested inner PCR (performed with IU50 [5'-GGAACTACTGTCTTCACGCAGA-3' {nt 51 to 72}] and P1) was carried out on 5 µl of the outer PCR mix under the same cycling conditions as above except with an initial denaturation temperature of 94°C for 5 min and an annealing temperature of 60°C. PCR products (4 µl) were electrophoresed in a 1% agarose gel and visualized by ethidium bromide staining. During PCR, multiple negative controls without template DNA were included. To avoid contamination, separate rooms were used for mixing of reagents, addition of sample, and PCR analysis.

Cooperative oligonucleotides in capture of HCV rRNA. (i) Preparation of HCV rRNA. To allow for generation of a 649-nt transcript by in vitro transcription, purified plasmids containing the 5' NTR of HCV (genotypes 1a, 2b, and 3a) were digested with NarI (258 bp downstream of the insert) and the resulting linearized DNA was precipitated and dissolved in 50 µl of diethylpyrocarbonate (DEPC) (Sigma Chemical Co.)-treated H₂O. Transcription from the T7 promoter was performed on 1.5 µg of this linearized DNA, as previously described (24). After transcription, template DNA was fragmented by restriction digestion (with AvaI) and treatment with 8 U of RNase-free DNase I (Boehringer) at 37°C for 45 min. Following phenol-chloroform extraction and ethanol precipitation, the resulting pellet containing HCV recombinant RNA (rRNA) was resuspended in 50 µl of DEPC-treated H₂O. A fivefold dilution series in 0.2 µg of yeast RNA per µl was made and stored at 70°C.

(ii) Analysis of HCV rRNA by biosensor. Biosensor experiments were performed with a BIAcore 2000 instrument (Biacore, Uppsala, Sweden), as described previously (16). Briefly, a biotinylated capture oligonucleotide with a sequence identical to that of C1 was immobilized on the sensor chip precoated with streptavidin (Sensor chips SA; Biacore) to a level of approximately 500 to 1,000 RU (1,000 RU corresponds to approximately 1 ng/mm² [21]). Six microliters of nondiluted rRNA transcripts was prehybridized to 0.5 µM probe (P1) in 100 µl of 6× SSPE by incubation at 54°C for 15 min, and 40 µl of these hybridization mixes were injected over the immobilized biotinylated capture oligonucleotide. Samples with no prehybridizing probe were treated in exactly the same manner. A pulse of 50 mM NaOH was used to regenerate the surface. This experiment was repeated for statistical analysis of the capture of rRNA. One flow cell without immobilized oligonucleotide was used as a control surface.

(iii) Magnetic-bead capture and detection of HCV rRNA by RT-PCR. The procedure for capturing rRNA on beads is essentially the same as that outlined for the capture of rDNA, namely, prehybridization of probe to 30 µl of rRNA at 54°C for 15 min, followed by capture onto magnetic beads (coupled with the HCV-specific capture probe C1) by rotation at room temperature for 90 min. The beads with the captured rRNA were washed three times in 100 µl of B/W buffer, washed twice in 100 µl of 10× PCR buffer (they were changed to a new microcentrifuge tube prior to the final washing step), and resuspended in 100 µl of DEPC-treated H₂O. If the bead suspension was not used immediately for RT-PCR, it was stored at 70°C. During transcription and RNA capture, all glassware and solutions (with the exception of Tris buffers) were DEPC treated to avoid possible contamination with RNases. Solid-phase RT and outer PCR were performed in a one-tube format with the OU49 and P1 primers. RT was carried out on 5 µl of resuspended beads at 37°C for 1 h (with continuous rotation) by using 0.5 U of MMLV Reverse Transcriptase (Pharmacia Biotech, Uppsala, Sweden) (with the prehybridizing probe [P1] as the RT primer), followed by PCR amplification with 2 U of AmpliTaq Gold (Perkin-Elmer) in a total reaction volume of 50 µl. The reaction conditions were the same as those described above for the outer PCR, with the addition of a PCR preheating step at 94°C for 12 min to activate AmpliTaq Gold. Four micrograms of yeast RNA was also included to prevent inhibition of Taq DNA polymerase activity by reverse transcriptase (18). Positive and negative controls were included, as well as a "no reverse transcriptase" control. Seminested inner PCR was carried out as described above.

Cooperative oligonucleotides in capture of HCV RNA from clinical samples. Serum samples stored at 20°C from HCV-infected patients were used. Initially, two HCV-positive samples (genotype 1a) were, in parallel, quantitated with the Amplicor HCV Monitor test (Roche Molecular Systems) and serially end diluted in a fivefold fashion in HCV-negative serum. One hundred microliters of these serum samples were then prehybridized with 0.5 µM probe (P1) in 1 ml of 6× SSPE containing 1 µg of Escherichia coli tRNA (Boehringer) and 500 µl of solution D (4 M guanidinium thiocyanate, 25 mM sodium citrate [pH 7], 0.5% sarcosyl, 0.1 M -mercaptoethanol) by heating at 60°C for 10 min followed by rotation at room temperature for 45 minutes. Magnetic beads (250 µg) covalently coupled with C1 (prepared as described above) were then added to the hybridization mix and rotated at room temperature for 1 h to facilitate capture. The beads were then washed four times in 100 µl of B/W buffer and twice in 100 µl of 1× PCR buffer. The beads were resuspended in 20 µl of H₂O, heated to 70°C for 3 min, and placed immediately on ice. RT-PCR was carried out on 10 µl of the suspension for 35 cycles, as described previously (26), with AmpliTaq DNA polymerase and the primers OU49 and OD66 (OD66 was used as the RT primer). Inner PCR was carried out for 35 cycles on 5 µl of the outer PCR product with the primers IU50 and ID56 (5'-TCGCAAGCACCCTATCAGGCAG-3' [nt 289 to 310]). A further nineteen HCV-positive serum samples, quantified with the Amplicor Monitor test and genotyped as described by Okamoto et al. (15), were then retrospectively evaluated by this capture method (with every sixth sample being a non-HCV serum negative control).

	RESULTS

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Cooperative oligonucleotides in magnetic-bead capture of an HCV rDNA fragment. In a previous study, using analytical biosensor technology, we analyzed the capture of single-stranded rDNA targets when prehybridizing oligonucleotides adjacent to the capture probe were used. Here, cooperative oligonucleotide modules were investigated for preparative purposes (employing magnetic bead technology) with a model system similar to that described previously (16) for the analysis and detection of HCV (Fig. 1). Single-stranded rDNA corresponding to the 5' NTR of HCV (genotypes 1a, 2b, and 3a) was prepared by PCR amplification and was then fivefold serially end diluted. The solid support for the first set of experiments was magnetic beads covalently coupled to an 18-mer probe (C1) complementary to the virus target. As illustrated in Fig. 1, an oligonucleotide module (P1) designed to anneal adjacent to C1 was prehybridized to the serially diluted templates at 54°C for 15 min. The hybridization mixtures were subsequently incubated with magnetic beads (at room temperature for 90 min) to facilitate solid-phase capture. Samples without the prehybridizing probe were prepared in parallel. All samples were tested in duplicate, and a control bead sample (no DNA) was included to monitor any cross-contamination during the washing steps, etc. After incubation, the beads were washed and transferred to PCR tubes containing reagents and primers for outer PCR amplification of the HCV target region. A seminested inner PCR was carried out to allow for comparison at the PCR plateau level, at which all dilutions have reached saturation irrespective of the initial copy number (20, 24). This is illustrated by the roughly equal intensity of the PCR fragments after gel electrophoresis. One set of results is depicted in Fig. 2 and shows that when the prehybridizing probe is included, an approximate fivefold increase in sensitivity is achieved (Fig. 2A) compared to capture without the prehybridizing probe (Fig. 2B). The control beads (without target DNA) were negative after PCR. In addition, capture with magnetic beads (and a prehybridized probe) showed equal sensitivity to PCR performed directly on the 5' NTR dilution series (Fig. 2C), indicating no loss of target during capture. A positive control was included to monitor the PCR efficiency, while reagent controls monitored the PCR solutions and cross-contamination.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the oligonucleotide-assisted capture method. The oligonucleotide module (P1) is initially prehybridized to the HCV target at elevated temperatures for 15 min, and this hybridization complex is then captured with the immobilized capture probe. Immobilization of the capture probe on a chip surface facilitates analysis by biosensor, while coupling of the capture probe to magnetic beads allows HCV detection by PCR.

View larger version (71K): [in this window] [in a new window]   FIG. 2.   Seminested PCR (generating a 273-bp fragment). (A) rDNA dilution series after capture with the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions of 57 to 510); lane 5 corresponds to amplification of the control beads (no DNA). (B) rDNA dilution series after capture without the prehybridizing probe. Lanes 1 to 4 correspond to dilutions 1 to 4 (dilutions 57 to 510); lane 5 is a PCR-negative control. (C) rDNA dilution series prior to capture. Lanes 1 to 5 correspond to PCR products derived from amplification of fivefold dilutions of HCV single-stranded rDNA (dilutions of 57 to 511). Bacteriophage  restricted with PstI was used as a marker (M).

Cooperative oligonucleotides in capture of an HCV rRNA fragment. (i) Biosensor analysis of capture of HCV rRNA. To allow a better comparison with clinical samples, in vitro-transcribed RNA corresponding to genotypes 1a, 2b, and 3a was generated for use initially in analytical BIAcore experiments. The BIAcore measures changes in the mass of molecules bound to the surface by the principle of surface plasmon resonance (7). These changes are measured in real-time and are presented in a sensorgram as resonance units (RU) versus time. The solid support in these experiments was a streptavidin chip with a biotinylated capture probe (identical in sequence to C1) immobilized on the chip surface. The cooperative oligonucleotide module (P1) which anneals adjacent to the immobilized capture probe site was prehybridized to the 649-nt rRNA transcript at 54°C for 15 min, and the complex was then injected over the chip surface (Fig. 3, schematic array 1). A control sample without the prehybridized probe was processed in parallel (Fig. 3, schematic array 2). A representative result from rRNA (genotype 2b) is presented as an overlay plot (Fig. 3), and the data clearly illustrates that significantly more target rRNA is captured when a prehybridizing probe has been employed. It is also important to note that the reactions have not reached saturation during the injection pulse (20 min); therefore, it is likely that the absolute differences are even higher. To investigate if these responses were reproducible, statistical analysis of the capture of rRNA (genotypes 1a, 2b, and 3a) was performed. Each genotype was analyzed five times, and the resulting coefficients of variation (CVs) ranged from 6.2 to 6.8%.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   BIAcore analysis of the oligonucleotide-assisted capture of rRNA. An overlay plot of processed sensorgrams (generated by subtraction of the responses from a control surface) shows the capture of in vitro-transcribed HCV RNA with and without the prehybridizing probe (P1).

(ii) Magnetic-bead capture of HCV rRNA. As a result of the successful BIAcore analyses, the rRNA model system was further evaluated for preparative purposes on magnetic beads with a covalently bound capture probe (C1). To facilitate comparison of capture with and without the cooperative oligonucleotide, the target rRNA was fivefold serially diluted and captured in duplicate, as described above for the rDNA template. After a washing step, one-tube RT-PCR was performed on the samples followed by seminested inner PCR. A representative result is presented in Fig. 4A and B, which show that when the prehybridized probe was used, the sensitivity was improved approximately fivefold. However, repeated analysis of this diluted rRNA target occasionally showed up to a 25-fold improvement in capture with the prehybridization probe. We believe that this observed variation is not a result of differences in capture efficiency but rather reflects stochastic variation within end-diluted rRNA specimens (24). To further dissect the differences shown in Fig. 4A and B and to exclude the possibility that the amplified fragment corresponding to the 5⁹ dilution was a spurious PCR product, a twofold dilution series of the captured rRNA (with and without the prehybridized probe) corresponding to the 5⁸ dilution was amplified, confirming at least a fourfold increase in sensitivity when a prehybridization probe was included (Fig. 4C and D).

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Seminested RT-PCR (generating a 273-bp fragment) of in vitro-transcribed HCV rRNA after capture with (A) and without (B) the prehybridizing probe (lanes 1 to 6 correspond to dilutions 1 to 6 [dilutions of 56 to 511]). Also shown is seminested RT-PCR (generating a 273-bp fragment) of a twofold dilution series of dilution 3 (i.e., dilution of 58) after capture with (C) and without (D) the prehybridizing probe (lanes 1 to 4), with lanes 1 corresponding to dilution 3. Bacteriophage  restricted with PstI was used as a marker (M).

Extraction of HCV RNA from serum samples with cooperative oligonucleotides and magnetic beads. The results with our two model systems (rDNA and rRNA), which indicated that cooperative oligonucleotide modules improve solid-phase capture, led us to evaluate this approach on HCV-positive serum samples. However, in contrast to the model systems, where the target is <700 nt, the target here is 9,600 nt.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Outer RT-PCR (A and B) and nested PCR (C and D) of a fivefold dilution series of HCV serum. Samples were amplified after capture with (A and C) and without (B and D) the pre-hybridizing probe. Lanes 1 correspond to the original serum sample; lanes 2 to 8 are fivefold serial dilutions of the original serum sample. Lanes 9 are PCR-negative controls. The outer PCR product is 324 bp, and the inner PCR product is 260 bp. The additional fragment visible after nested PCR corresponds to the outer PCR product. The marker (M) is X174-RF DNA digested with HaeIII.

	DISCUSSION

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Since the detection of HCV is by necessity dependent upon nucleic acid preparation protocols, the method of RNA extraction is a critical factor in the optimization of such diagnostic assays. Indeed, the method selected for RNA extraction can have a profound effect on the sensitivity of subsequent RT-PCR assays (14, 25). The guanidinium thiocyanate method described by Chomczynski (3) is among the most sensitive and reproducible protocols for extraction of viral RNA (25), but the phenol-chloroform-isoamylalcohol extractions and ethanol precipitation steps make simultaneous processing of multiple samples difficult. To simplify the RNA extraction protocol, thereby facilitating automation, capture methods with magnetic beads have been developed (1, 5, 13, 17, 23). However, these capture assays often show losses in sensitivity in comparison to the standard protocols. The HCV capture protocol of van Doorn et al. (23) was 10-fold less sensitive than that of a standard protocol, while Hsuih et al. (5) lost 60% of the HCV RNA target when they used two independent capture probes to extract RNA.

In this study, we present a novel approach for improving the sensitivity of nucleic acid capture assays while still taking advantage of the effective guanidinium thiocyanate reagent. From previous studies, it was noted that adjacent oligonucleotides interact cooperatively both in solution (8, 11) and in a solid-phase mode in which one oligonucleotide is immobilized and the other is prehybridized to the target (16).

The latter approach has allowed us to design efficient sample preparation systems for HCV. We have shown in a comparative study that the prehybridizing probe increases the capture efficiency for two types of recombinant HCV targets (DNA and RNA). This also holds true for clinical samples, for which all genotypes tested were captured, although more samples have to be analyzed, especially in view of the sample (genotype 3a) which showed only a minor improvement in capture efficiency when the cooperative oligonucleotide was used (Table 2). Preliminary results from the capture of human immunodeficiency virus type 1 (pol region) have also shown significant improvement in capture efficiency when a cooperative oligonucleotide is employed.

From previous experiments using the biosensor instrument, we calculated that the capture of single-stranded DNA was reproducible (CV, 2.6%) (16). Here we have followed up these results by investigating the reproducibility of the capture of rRNA. The calculated CVs are in the range of 6.2 to 6.8%, which further supports the robustness of this method. The slightly higher CVs for rRNA targets may be due to degradation of the rRNA, since there was at least a 3- to 4-h interval between injections of the first and last sample.

The bDNA data further supports our claim of increased sensitivity when the prehybridization probe is included, as these results clearly show a consistent improvement in capture efficiency. This final experiment is especially significant since it shows the comparison of the conventional extraction protocol with our magnetic bead system. Our capture assay extracts RNA of different genotypes and compares well to conventional extraction procedures. Furthermore, it has the advantage of being a simple approach, enabling the design of automated systems. Also, we believe that the use of a selective extraction method in which nonspecific RNA is removed will allow for a greater sensitivity in detection, as the high total RNA concentration isolated by conventional extraction methods may inhibit PCR amplification (13).

In this study, we used probes and PCR primers complementary to the most conserved region of HCV, the 5' NTR, so that all HCV subtypes were readily captured. A recently identified alternative target (a 98-bp region 3' of the NTR) (10), which is strongly conserved among all genotypes, may be an even better target for probe capture. This region is predicted to form three very stable stem-loop structures; therefore, a prehybridized oligonucleotide module could be particularly useful in a capture method directed toward this region. Magnetic-bead-mediated sample preparation will also facilitate RT-PCR of the whole genome by minimizing the risk of shearing the RNA template. This is important when longer RT-PCR products are desired, as the purity and integrity of the RNA template are critical factors in the success of long-range RT-PCR (22).

Preliminary studies have suggested that the protocol for virus capture could be shortened by combining prehybridization, sample lysis (in guanidinium thiocyanate), and bead capture in a single step. Thus, only a simple washing step would be required prior to RT-PCR, simplifying the procedure even further. However, a more thorough study with additional clinical samples needs to be performed to validate our results. Taken together, we have developed a new capturing strategy which may be useful in the development of diagnostic systems for other single-stranded targets.