Development of magnetic capture hybridization and quantitative polymerase chain reaction for hepatitis B virus covalently closed circular DNA

Abstract

Background: Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) served as a vital role in the life cycle of the virus and persistent infection. However, specific and quantitative methods for cccDNA detection have not been available.

Objectives: Our aim was to develop and primarily evaluate a quantitative method for HBV cccDNA based on magnetic capture hybridization and quantitative PCR technology.

Materials and methods: The functionalized-nanoparticles specifically to capture HBV cccDNA, located on both sides of relaxed circle DNA (rcDNA) gap, were designed. Then, magnetic capture hybridization and quantitative PCR (MCH-qPCR) assay were developed and its performance was primarily evaluated with cccDNA standards and serum samples of patients with chronic hepatitis B.

Results: Specific nanoparticles of cccDNA capture were prepared and a magnetic capture hybridization and quantitative assay method for cccDNA was developed successfully. The limit of detection was 90 IU/mL, and a good linear relationship in the range of 10(2)-10(6) IU/mL was revealed (r(2) = 0.994) with the MCH-qPCR. Compared with directly real-time PCR, a high content of HBV DNA did not affect the detection of cccDNA for the MCH-qPCR method, and there was no cross-reactivity between cccDNA and rcDNA.

Conclusions: The novel MCH-qPCR method has good sensitivity and specificity. It could meet the requirement of clinical routine detection.

Keywords: Hepatitis B Virus; Magnetic; PCR Technology.

Figure 1.. TEM Photograph of the Fe₃O₄ Nanopowders (Left) and Silica-Coated Nanopowders (Right)

Particle sizes of single-phase nanopowders (Fe₃O₄) were between 8 and 15 nm, average size was about 13.2 nm, and that of silica-coated nanoparticles ranged from 65 to 93 nm, its average size was about 84 nm. Surface modification of nanoparticles (silica-coated nanoparticles) could greatly reduce aggregation.

Figure 2.. The Magnetic Hysteresis Loop of the Nanoparticles (Left) and FITR Pattern of the Silica-Coated Nanoparticles (Right)

Left, the saturation magnetization of the nanoparticles was approximately 55 emu/g, lower than the saturation magnetization of bulk magnetite (92 emu/g). Moreover, non-hysteresis and remanence phenomena were observed, which indicated an obvious superparamagnetic phenomenon that magnetism only existed when the external magnetic field was present; when the external magnetic field was removed the magnetic field completely disappears. Right, The Fe-O-Fe stretching vibration was 471/cm, and the stretching vibration of Si-O was 1104/cm. Therefore, the main peaks show that the synthetic particles contained Fe₃O₄ and silica.

Figure 3.. Absorbance Curves of the Supernatant of Magnetic Nanoparticles Modified by Streptomycin Coupled With the Biotin-Labeled Probe

A, Absorbance curve of the supernatant when only the probe was added; B, Absorbance curve of the supernatant after the coupling reaction; c. Absorbance curve of the first washing solution (PBS) after the coupling reaction.

Figure 4.. Fluorescence of the Supernatants Containing Different Concentrations of Nanoparticle Probes Hybridized With the Complementary FITC-Labeled Probes

A, blank; B, control group; C, 200 pmol of probes; D, 150 pmol of probes, E; 100 pmol of probes; and F, 50 pmol of probes

Figure 5.. Linear and Regression Analyses Between Expected Values of cccDNA Concentration and Values Determined by MCH-qPCR

Line-a was the curve fitted between the actual added concentration (log10) and the expected concentration (log10), y = x, R² = 1; Line-b was the curve fitted between the concentration determined (log10) by MCH-qPCR and the expected concentration (log10).

Figure 6.. Specificity Experiment for MCH-qPCR

Recovery (%): Group 10⁶ = (93.2 ± 2.0); Group 10⁵ = (94.1 ± 1.2); Group 10⁴ = (90.1 ± 3.4); Group 10³ = (90.8 ± 3.8); Group 10² = not detected; control (HBV DNA) = not detected. The raw data of all groups (detected) were multiplied by the dilution factor (10-fold). The differences among groups 10⁶, 10⁵, 10⁴ and 10³ were not significant (P > 0.05).