Automated Specific Capture of Hepatitis C Virus RNA with Probes and Paramagnetic Particle Separation

Abstract

We developed and evaluated a prototype automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation. HCV RNA was isolated from serum by lysis of virus particles with a chaotropic agent, followed by hybridization of the RNA with biotinylated probes and capture of the hybridized RNA with streptavidin-coated paramagnetic particles. After washing of the hybrid-particle complexes to remove nonspecifically bound materials, the particles were resuspended in a specimen diluent and were then ready for amplification and detection with a fully automated PCR system (COBAS AMPLICOR; Roche Diagnostic Systems). The analytical sensitivity in the dilution series was 33 copies per ml or greater. Comparison of the test results with those obtained by a manual method based on organic extraction and precipitation of RNA (SepaGene RV-R; Sanko Junyaku Co., Ltd.) showed 93% (49 of 53 samples) sensitivity and 100% (12 of 12 samples) specificity. There was 94% overall agreement between results. When RNA was extracted by the manual method from serum containing 10 3 or 10 5 copies of HCV per ml in the presence of heparin, there was an inhibitory effect on detection of both HCV RNA and the internal control. In contrast, when RNA was extracted from the serum by the automated method, there was no inhibitory effect. This inhibitory effect of heparin on the manual method was also observed for a series of serum specimens from a hemodialysis patient, but the inhibitory effect was eliminated by the automated specimen preparation method. In summary, a fully automated RNA extraction system for PCR detection of HCV RNA by use of specific capture with probes and magnetic bead-fluid separation was shown to have performance similar to that of the conventional manual method. In addition, it successfully eliminated the inhibitory effect of the heparin in the serum and permitted the detection of HCV RNA in serum samples from a hemodialysis patient. The prototype automated RNA extraction system is suitable as a totally automated system, starting with RNA extraction to detection of HCV, if it was combined with the fully automated COBAS AMPLICOR PCR system.

Functional Magnetic Nanoparticles for Biodefense and Biological Threat Monitoring and Surveillance

“…Different approaches were developed to enrich samples with a germ-specific DNA and thus improve sensitivity and specificity of germ identification by PCR. In some cases, prior to PCR analysis, DNA was extracted from soil or water samples using particles with attached DNA-capture fragments complementary to the target DNA. ,,− However, the high specificity of such DNA-specific particles toward a single pathogen is a significant limitation for the surveillance of multiple germs, which calls for large arrays of the DNA sequences specific to organisms to be surveyed. In addition, particles modified with DNA capture probes are not stable, because of fast DNA degradation in the environment.…”

Microparticle-Based Detection of Viruses

“…Furthermore, magnetic microparticles were previously exploited as a pretreatment step for nucleic acid amplification of flaviviruses. Miyachi et al extracted HCV RNA from serum samples using hybridized magnetic microparticles, allowing target sequences to be isolated from the molecules that could inhibit amplification [ 74 ]. Recent work has combined magnetic microparticles with AuNPs to create antibody-sandwich complexes around HCV core antigens ( Figure 4 a) [ 75 ].…”

Automated Extraction of Plant Viral RNA Using the Nucleic Acid Instrument Based on Magnetic Nanobeads

“…Furthermore, magnetic separation with high efficiency is especially suitable for large-scale purification [9,10] and automation [11]. As early as 1999，Hayato Miyachi.etc developed and evaluated an automated specimen preparation instrument for the specific capture of hepatitis C virus (HCV) RNA with probes and magnetic bead-fluid separation [12]. Subsequently, there were many reports about automated nucleic acid extraction [13,14,15].…”