A Highly Sensitive Diagnostic System for Detecting Dengue Viruses Using the Interaction between a Sulfated Sugar Chain and a Virion

Abstract

We propose a novel method of detecting trace amounts of dengue virus (DENVs) from serum. Our method is based on the interaction between a sulfated sugar chain and a DENV surface glycoprotein. After capturing DENV with the sulfated sugar chain-immobilized gold nanoparticles (SGNPs), the resulting complex is precipitated and viral RNA content is measured using the reverse-transcription quantitative polymerase chain reaction SYBR Green I (RT-qPCR-Syb) method. Sugar chains that bind to DENVs were identified using the array-type sugar chain immobilized chip (Sugar Chip) and surface plasmon resonance (SPR) imaging. Heparin and low-molecular-weight dextran sulfate were identified as binding partners, and immobilized on gold nanoparticles to prepare 3 types of SGNPs. The capacity of these SGNPs to capture and concentrate trace amounts of DENVs was evaluated in vitro. The SGNP with greatest sensitivity was tested using clinical samples in Indonesia in 2013-2014. As a result, the novel method was able to detect low concentrations of DENVs using only 6 μL of serum, with similar sensitivity to that of a Qiagen RNA extraction kit using 140 μL of serum. In addition, this method allows for multiplex-like identification of serotypes of DENVs. This feature is important for good healthcare management of DENV infection in order to safely diagnose the dangerous, highly contagious disease quickly, with high sensitivity.

Fig 1. Binding affinities of dengue viruses (DENVs) for various sugar chains immobilized on an array-type Sugar Chip.

The map of immobilization, an example of the data on the binding, and the list of sugar chains are shown in S1 Fig. DENVs re-suspended in PBS-T at the concentration of 1.5–12 μg/mL (total viral protein) were loaded onto the sugar chain chip at a flow rate of 150 μL/min, and the signal data (brightness, recorded by a CCD camera, indicating the binding affinity) were collected by the manufacturer’s analytical software (Toyobo). The signal intensities of 8 independent data points for each spot were averaged and plotted on the Y-axis.

Fig 2. The concentration procedure for dengue virus (DENV) virions using sulfated sugar chain-immobilized gold nanoparticles (SGNPs).

A DENV solution was diluted 1:10⁴ or 1:10⁶ in PBS. PPT, precipitate obtained from the treatment with SGNP; SUP, supernatant obtained from the treatment with SGNP; No SGNP, sample without treatment with SGNP. T_m for DENV-1 was 85 ± 0.3°C. Upper: fluorescent intensity vs. cycle number (Ct); Lower: melting curve.

Fig 3. Melting curves for dengue virus (DENV) genotypes 1 through 4 using real-time PCR with SYBR Green I.

The peaks of melting curves were clearly separated. T_m values for DENV-1, DENV-2, DENV-3, and DENV-4 were 84, 81.5, 85, and 83°C, respectively.