Rapid functionalisation and detection of viruses via a novel Ca²⁺-mediated virus-DNA interaction

Abstract

Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool.

Virus detection and identification in minutes using single-particle imaging and deep learning

“…For SARS-CoV-2 imaging, the cationic labelling solution was buffered with 20 mM Tris, pH 8. Virus labelling with CaCl2 has been described previously (9); SrCl2 provides similar results (Fig. 1).…”

“…3). The labelling is an electrostatic interaction between the phosphate backbone of the DNA and the lipid membrane of the virus (9). Different viruses will therefore exhibit differences in labelling efficiency and coverage due to their different isoelectric points (the pH at which a virus has a neutral surface charge), e.g.…”

“…Laboratory grown virus strains and DNAs. The influenza strains (H1N1 A/Puerto Rico/8/1934 (PR8), H3N2 A/Udorn/72 (Udorn), H1N1 A/WSN/33 (WSN) and H3N2 A/Aichi/68 (X31)) used in this study have been described previously (9). Briefly, WSN, PR8 and Udorn were grown in Madin-Darby bovine kidney (MDBK) or Madin-Darby canine kidney (MDCK) cells and X31 was grown in embryonated chicken eggs.…”

“…Single-stranded oligonucleotides labelled with either red or green dyes were purchased from IBA (Germany). We have previously characterised a range of DNAs of different length and sequence and shown that as long as the DNA is longer than 20 bases robust labelling occurs, regardless of sequence (9). The 'red' DNA used in this manuscript was modified at the 5' end with ATTO647N ( Specimens were maintained in Copan Universal Transport Medium (UTM) before being inactivated in 4% final concentration of formaldehyde (Pierce) for 30 minutes at room temperature, or 1% formaldehyde for 5 minutes at room temperature for the 104 samples used in the second clinical trial (21).…”

Label-free virus-antibody interaction monitoring in real time by common-path interferometry

“…For virus particles, fluorescent labels have been mostly used to tag the capsid protein [6] or to tag DNA viral genomes [7]. To note that a rapid virus detection method has been reported requiring about one minute to obtain a result, in which calcium labeling has been used to track the mobility of viruses when interacting with specific antibodies [8]. However, the optimization of the labeling process is crucial in order to retrieve high contrast images and yet, fluorescence labels suffer from phototoxicity, photobleaching, photostability, and saturation.…”