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.
Highlights
Dengue viruses (DENVs), which have 4 phylogenetically and antigenically distinct types (DENV-1 to dengue virus (DENV)-4), are emerging arthropod-borne flaviviruses that cause dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS), mostly in tropical and subtropical countries [1]
The DENVs used in this study strongly and bound to chondroitin sulfate type-E (CS-E), heparin (Hep), low molecular weight (LMW) dextran sulfate (DS25), synthetic sulfate partial disaccharides in heparin or chondroitin sulfate
We applied our glyco-nanobiotechnology techniques to improve the detection of DENV at very low concentrations in serum samples
Summary
Dengue viruses (DENVs), which have 4 phylogenetically and antigenically distinct types (DENV-1 to DENV-4), are emerging arthropod-borne flaviviruses that cause dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS), mostly in tropical and subtropical countries [1]. Co-occurrence of multiple types may lead to an increase in the number of severe dengue cases. Current DENV diagnostic methods are based on serological detection of circulating antibodies. Specificity of these tests suffer due to cross-reactivity from antibodies against related flaviviruses, they are insufficient. Alternative methods for the detection of DENV rely on the measurement of viral RNA using reverse transcription polymerase chain reaction (RT-PCR). We describe a novel method to capture and concentrate DENVs by sugar chain-immobilized gold nanoparticles (SGNP), followed by PCR-based detection. We compared our novel SGNP method with a commercially available RNA extraction kit to detect DENV in patient serum and plasma samples in a clinical setting using reverse-transcription quantitative PCR (RT-qPCR)
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