A Highly Sensitive Strategy for Low-Abundance Hepatitis B Virus Detection via One-Step Nested Polymerase Chain Reaction, Chemiluminescence Technology and Magnetic Separation

Hepatitis B virus (HBV) has been always threatening to health. It is significant to establish an effective strategy for HBV carriers and hepatitis B patients diagnosis at early stage of infection in clinic. Herein, we combined nested PCR, chemiluminescence assay and magnetic separation technology to establish a highly sensitive and stable strategy for low-abundance HBV-DNA detection. Six types of monodisperse Fe3O4 magnetic beads (MBs) were fabricated and coated with SiO2 layer (MBs@SiO2). The effect of MBs@SiO2 size on HBV-DNA extraction and chemiluminescence assay was explored, and results displayed that the MBs@SiO2-F particles with largest size were much more suitable for HBV-DNA extraction than others, and MBs@SiO2-C with the size range between 330 nm-450 nm performed best in chemiluminescence assay. As one of key factors, the concentration (or density) of the probe fixed on MBs@SiO2 surface was optimized for chemiluminescence assay, exhibiting the best assay result when reaction system contained 2.0 mu M of probe. Finally, under the best reaction conditions, low-abundance HBV serum specimens were sensitively and stably detected, and the result of HBV specimen with 34 copy/mL loads showed significant difference with negative and blank (p < 0.001).