Reduction in the viability of FCV and MNV after contact with ethanol.

<p>Virus reduction was observed after 20 s (A) or 30 s (B) contact time with a commercial ABHR (62% ethanol) and an aqueous solution of 75% (v/v) ethanol on the fingerpads of adult subjects (n = 6). Significant difference between FCV and MNV (p<0.01) indicated by asterisks.</p

Reduction of MNV after 20 second contact with ethanol.

<p>MNV reduction was observed after 20 s contact time with one of either a commercial ABHR (62% ethanol), 75% ethanol or 80% aqueous ethanol solution on the fingerpads of adult subjects (n = 6). Significant differences were observed between both 62% and 75% (p<0.01) and 80% (p<0.01) indicated by asterisks (*). A significant difference was also seen between 75% and 80% (p<0.01) indicated by the double dagger (‡).</p

Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology

<div><p>DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.</p></div

Binding assays.

<p>A) Binding affinity measurements for 5′-fluorescein-modified AG3 with MNV using a polycarbonate filter binding assay (diamonds) and a fluorescence anisotropy assay (triangles). The binding affinity of 5′-fluorescein tagged AG3 to FCV (circles) as well as a non-specific DNA control with MNV (squares) were also measured by anisotropy. Measurements are fitted using either the Hill or Logistic functions (solid lines). Inset: Much higher concentrations of virus are required in order to show any anisotropy change in the controls. B) Fluorescence anisotropy results from binding of 5′-fluorescein-modified AG3 with varying concentrations of GII.3 HuNoV capsid.</p

Schematic diagram of the electrochemical detection protocol adopted in this study.

<p>A thiolated norovirus-specific DNA aptamer was self-assembled onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Binding of the virus to the immobilized aptamer causes a decrease in the redox current, measured <i>via</i> square wave voltammetry.</p

Aptasensor results.

<p>(A) Cyclic voltammograms of the norovirus aptasensor after each immobilization or binding step. The [Fe(CN)₆]^3−/2− redox couple was monitored for these experiments and cyclic voltammograms were recorded at a scan rate of 100 mV s^,–1 where (<i>a</i>) bare SPGE; (<i>b</i>) after self-assembly of the thiolated norovirus specific aptamer; (<i>c)</i> after back-filling with 1 mM 2-mercaptoethanol. (B) Square wave voltammograms obtained using (<i>a</i>) 20 aM, (<i>b</i>) 40 aM, (<i>c</i>) 60 aM, (<i>d</i>) 80 aM, (<i>e</i>) 100 aM, and (<i>f</i>) 120 aM of norovirus in buffer. (C) Calibration plot of current <i>vs.</i> concentration of norovirus. (D) Selectivity experiments performed using (a) buffer alones, (b) 5000 PFU of vesicular stomatitis virus, (c) 5.1 mg mL⁻¹ HSA, (d) 5000 PFU of vaccinia virus (e) 120 aM of norovirus. All experiments were performed in Dulbecco’s phosphate buffered saline after incubation with the developed aptasensor for 1 hr at 25°C. Square wave voltammograms were carried out in the range of −400 to 800 mV with a step potential of 4 mV, amplitude of 5 mV and frequency of 10 Hz. Electrochemical measurements were performed in 25 mM phosphate buffer (pH 7), containing 4 mM K₃[Fe(CN)₆] and 10 µM hexaamine ruthenium chloride.</p

Summary of rounds for MNV SELEX.

<p>Target stringencies are outlined for sets of rounds (VP = viral particles). Retained fractions were assessed by fluorescence. DNA retained after counter-selection rounds (8 and 9) represents pool that did not interact with the indicated targets (FCV = feline calicivirus and DMEM = dialyzed components of Dulbecco’s Modified Eagle Medium).</p

Summary of aptamer candidates.

<p>Summary of aptamer candidates.</p

Eigenvectors from principal component analysis of plasma cytokine levels of pooled Caucasians and First Nations individuals.

<p>Eigenvectors from principal component analysis of plasma cytokine levels of pooled Caucasians and First Nations individuals.</p

Characteristics of participants.

<p>Data are expressed as means ± standard deviation.</p><p>BMI, body mass index.</p><p>Significant difference between groups at.</p>*<p>p<0.05.</p