Cooperative oligonucleotides mediating direct capture of hepatitis C virus RNA from serum

Abstract

A novel method for direct capture of hepatitis C virus (HCV) RNA from clinical samples has been developed. This approach takes advantage of the cooperative interactions between adjacently hybridized oligonucleotides. Here, this cooperative effect was combined with solid-phase technology, whereby a capture probe was covalently coupled to magnetic beads and a second probe, which anneals adjacent to the capture probe site, was prehybridized in solution to the target. When these contiguously hybridized probes were used for the extraction of HCV RNA from clinical samples, the capture efficiency was increased up to 25-fold in comparison to capture with a single probe. The applicability of this sample preparation assay was further investigated by performing a comparative study with both a conventional guanidinium extraction method and a commercial quantitative assay.

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.

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 5⁻⁷ to 5⁻¹⁰); 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 5⁻⁷ to 5⁻¹⁰); 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 5⁻⁷ to 5⁻¹¹). Bacteriophage λ restricted with PstI was used as a marker (M).

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).

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 5⁻⁶ to 5⁻¹¹]). Also shown is seminested RT-PCR (generating a 273-bp fragment) of a twofold dilution series of dilution 3 (i.e., dilution of 5⁻⁸) after capture with (C) and without (D) the prehybridizing probe (lanes 1 to 4), with lanes 1 corresponding to dilution 3. Bacteriophage λ restricted with PstI was used as a marker (M).

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 φX174-RF DNA digested with HaeIII.