A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches

Abstract

BackgroundRecent epidemics of dengue viruses (DENV) coupled with new outbreaks on the horizon have renewed the demand for novel detection methods that have the ability to identify this viral pathogen prior to the manifestation of symptoms. The ability to detect DENV in a timely manner is essential for rapid recovery from disease symptoms. A modified lab-derived 10-23 DNAzyme tethered to gold nanoparticles provides a powerful tool for the detection of viruses, such as DENV.ResultsWe examined the effectiveness of coupling DNAzyme (DDZ) activation to the salt-induced aggregation of gold nanoparticles (AuNP) to detect dengue virus (DENV) progeny in mosquito cells. A DNAzyme was designed to recognize the 5’ cyclization sequence (5’ CS) that is conserved among all DENV, and conjugated to AuNPs. DDZ-AuNP has demonstrated the ability to detect the genomic RNA of our model dengue strain, DENV-2 NGC, isolated from infected Aedes albopictus C6/36 cells. These targeting events lead to the rapid aggregation of AuNPs, resulting in a red to clear color transition of the reaction mixes, and thus positive detection of the DENV RNA genome. The inclusion of SDS in the reaction mixture permitted the detection of DENV directly from cell culture supernatants without additional sample processing. Specificity assays demonstrated detection is DENV-specific, while sensitivity assays confirm detection at levels of 1 × 101 TCID50 units. These results demonstrate DDZ-AuNP effectively detects DENV genomes in a sequence specific manner and at concentrations that are practical for field use.ConclusionsWe have developed an effective detection assay using DNAzyme catalysis coupled with AuNP aggregation for the detection of DENV genomes in a sequence specific manner. Full development of our novel DDZ-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of DENV in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required.

Gold nanoprobe-based non-crosslinking hybridization for molecular diagnostics

“…AuNPs functionalized with DNA aptamers have been applied to the identification of DNA adducts as suitable biomarkers of exposure to a carcinogen agent, on a simple NCL-like system, where the presence of the analyte prevented aggregation of the aptamer-gold nanoprobes [77]. A different study reports AuNPs' derivatization with DNAzymes toward detection of viral RNA from dengue [79]. Upon hybridization of the DNAzyme-nanoprobe to the viral RNA, a DNAzyme-RNA duplex was formed at the nanoprobe surface, with a consequent stabilizing effect.…”

“…PCR is still considered the 'gold standard' technique for molecular detection associated with clinical diagnostics [78,79]. However, PCR-based techniques are generally more expensive due to reagents and enzymes, specialized personnel and equipment (see [78] and references therein).…”

State of diagnosing infectious pathogens using colloidal nanomaterials

“…Integrating plasmon coupling of GNPs with DNAzyme was studied to provide a linear amplification step and improve the sensitivity level in the detection of dengue virus nucleic acid. This system consists of DNAzyme attached to the surface of 15 nm GNPs, where DNAzyme gets triggered by the addition of magnesium ions for cleavage of viral RNA, and cleaved RNA induce aggregation of GNPs in the presence of salt and heat [162]. Another example includes the integration of the plasmon coupling of GNPs with multicomponent nucleic acid enzyme (MNAzyme) [163,164] (Fig.…”

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos

“…A técnica de AuNPs tem sido utilizada para diagnóstico de Mycobaterium tuberculosis (Hussain et al 2013), câncer de bexiga (Nossier et al 2014), Leishmania sp. (Andreadou et al 2014) e vírus da dengue (Carter et al 2013). A detecção do DNA é baseada na adsorção das bases nitrogenadas aos oligonucleotídeos e a superfície das AuNPs.…”

“…(Baptista et al 2006, Liandris et al 2009, Hussain et al 2013, Escherichia coli, com detecção de 54 nanogramas (ng) de DNA não amplificado e com 100% de sensibilidade (Bakthavathsalam et al 2012), RNA de vírus da dengue com detecção de 1×10 1 TCID50 unidade (Carter et al 2013) e Leishmania sp. com valores de detecção de 11,5ng à 50ng DNA e sensibilidade de 56% à 92% (Rosa et al 2013, Andreadou et al 2014.…”