Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings

The COVID-19 pandemic is challenging diagnostic testing capacity worldwide. The mass testing needed to limit the spread of the virus requires new molecular diagnostic tests to dramatically widen access at the point-of-care in resource-limited settings. Isothermal molecular assays have emerged as a promising technology, given the faster turn-around time and minimal equipment compared to gold standard laboratory PCR methods. However, unlike PCR, they do not typically target multiple SARS-CoV-2 genes, risking sensitivity and specificity. Moreover, they often require multiple steps thus adding complexity and delays. Here we develop a multiplexed, 1-2 step, fast (20-30 minutes) SARS-CoV-2 molecular test using reverse transcription recombinase polymerase amplification to simultaneously detect two conserved targets - the E and RdRP genes. The agile multi-gene platform offers two complementary detection methods: real-time fluorescence or dipstick. The analytical sensitivity of the fluorescence test was 9.5 (95% CI: 7.0-18) RNA copies per reaction for the E gene and 17 (95% CI: 11-93) RNA copies per reaction for the RdRP gene. The analytical sensitivity for the dipstick was 130 (95% CI: 82-500) RNA copies per reaction. High specificity was found against common seasonal coronaviruses, SARS-CoV and MERS-CoV model samples. The dipstick readout demonstrated potential for point-of-care testing in decentralised settings, with minimal or equipment-free incubation methods and a user-friendly prototype smartphone application. This rapid, simple, ultrasensitive and multiplexed molecular test offers valuable advantages over gold standard tests and in future could be configurated to detect emerging variants of concern

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection.

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity(1-5). The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions. Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures(1,6,7). We developed a new model(1) which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections

Self-Swabbing for Virological Confirmation of Influenza-Like Illness Among an Internet-Based Cohort in the UK During the 2014-2015 Flu Season: Pilot Study

BACKGROUND: Routine influenza surveillance, based on laboratory confirmation of viral infection, often fails to estimate the true burden of influenza-like illness (ILI) in the community because those with ILI often manage their own symptoms without visiting a health professional. Internet-based surveillance can complement this traditional surveillance by measuring symptoms and health behavior of a population with minimal time delay. Flusurvey, the UK's largest crowd-sourced platform for surveillance of influenza, collects routine data on more than 6000 voluntary participants and offers real-time estimates of ILI circulation. However, one criticism of this method of surveillance is that it is only able to assess ILI, rather than virologically confirmed influenza. OBJECTIVE: We designed a pilot study to see if it was feasible to ask individuals from the Flusurvey platform to perform a self-swabbing task and to assess whether they were able to collect samples with a suitable viral content to detect an influenza virus in the laboratory. METHODS: Virological swabbing kits were sent to pilot study participants, who then monitored their ILI symptoms over the influenza season (2014-2015) through the Flusurvey platform. If they reported ILI, they were asked to undertake self-swabbing and return the swabs to a Public Health England laboratory for multiplex respiratory virus polymerase chain reaction testing. RESULTS: A total of 700 swab kits were distributed at the start of the study; from these, 66 participants met the definition for ILI and were asked to return samples. In all, 51 samples were received in the laboratory, 18 of which tested positive for a viral cause of ILI (35%). CONCLUSIONS: This demonstrated proof of concept that it is possible to apply self-swabbing for virological laboratory testing to an online cohort study. This pilot does not have significant numbers to validate whether Flusurvey surveillance accurately reflects influenza infection in the community, but highlights that the methodology is feasible. Self-swabbing could be expanded to larger online surveillance activities, such as during the initial stages of a pandemic, to understand community transmission or to better assess interseasonal activity

Determining biosensing modes in SH-SAW device using 3D finite element analysis

Surface acoustic wave (SAW) sensors are electromechanical devices that exploit the piezoelectric effect to induce elastic (acoustic) waves which are sensitive to small perturbations: for example specific binding and recognition of disease biomarkers. Shear horizontal surface acoustic waves (SH-SAWs) are particularly suited to biosample analysis as the wave is not completely radiated and lost into the liquid medium (e.g., blood, saliva) as is the case, for example, in a device implementing Rayleigh waves. Here, using 3D finite element analysis (FEA) the nature of waves launched on a particular quartz device is investigated with respect to the cut of the quartz, the addition of gold guiding layers, and the addition of other linear elastic materials of contrasting acoustic properties. It is demonstrated that 3D FEA analysis showing the device's frequency shift with added guiding layer height reveals a proportional relationship in agreement with the Sauerbrey equation from perturbation theory. It is directly shown, given certain device parameters and a gold guiding layer, that shear horizontally polarized waves are launched on the surface with a dominant mode frequency around 250 MHz. This would be an appropriate biosensing mode in Point of Care (POC) testing for the particular properties of certain disease biomarkers delivered via a liquid medium

Differential stress induced by thiol adsorption on facetted nanocrystals

Polycrystalline gold films coated with thiol-based self-assembled monolayers (SAM) form the basis of a wide range of nanomechanical sensor platforms. The detection of adsorbates with such devices relies on the transmission of mechanical forces, which is mediated by chemically derived stress at the organic-inorganic interface. Here, we show that the structure of a single 300-nm-diameter facetted gold nanocrystal, measured with coherent X-ray diffraction, changes profoundly after the adsorption of one of the simplest SAM-forming organic molecules. On self-assembly of propane thiol, the crystal's flat facets contract radially inwards relative to its spherical regions. Finite-element modelling indicates that this geometry change requires large stresses that are comparable to those observed in cantilever measurements. The large magnitude and slow kinetics of the contraction can be explained by an intermixed gold-sulphur layer that has recently been identified crystallographically. Our results illustrate the importance of crystal edges and grain boundaries in interface chemistry and have broad implications for the application of thiol-based SAMs, ranging from nanomechanical sensors to coating technologies

A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings

Taking connected mobile-health diagnostics of infectious diseases to the field.

Mobile Health or mHealth - The application of mobile devices, their components and related technologies to healthcare is improving patients’ access to treatment and advice. Now, in combination with connected diagnostic devices it offers new possibilities to diagnose, track and control infectious diseases and improve health system efficiencies. In this context we look at these technologies and highlight their promise but also the challenges in realising their potential to increase patient access to testing, aid in their treatment and improve the capability of public health authorities to monitor outbreaks, implement responses, and assess the impact of interventions across the world

Spin-enhanced nanodiamond biosensing for ultrasensitive diagnostics

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness, low cost and selective manipulation of their emission. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity and frequency-domain analysis to separate the signal from background autofluorescence, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10−19 molar for a biotin–avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations

Tuneable Plasmonic Gold Dendrimer Nanochains for Sensitive Disease Detection

We report the development of a tuneable plasmonic nanochain immunoassay with increased sensitivity over traditional monodisperse nanoparticle lateral flow tests. Our approach takes advantage of the unique self-assembling properties of polyamidoamine dendrimers with gold nanoparticles in aqueous media to create one-dimensional nanochains, with a distinct red to blue colour change, attributable to a longitudinal plasmon resonance, which can be readily detected by eye and a digital camera. We optimise and characterise nanochain formation and stability using UV-visible spectroscopy, transmission electron microscopy and dynamic light scattering. As a proof-of-principle we focus on the application of nanochains for point-of-care diagnostics for p24, an important biomarker of early HIV infections and successfully detect p24 with a limit of detection of 5 ng ml−1 in pseudo-serum, 4 fold more sensitive than comparable studies with gold nanoparticles. These findings and underlying concepts highlight the potential of advanced functional organic–inorganic composite nanomaterials to diagnose infections, with broad applicability to non-communicable diseases

Decoupling competing surface binding kinetics and reconfiguration of receptor footprint for ultrasensitive stress assays

Cantilever arrays have been used to monitor biochemical interactions and their associated stress. However, it is often necessary to passivate the underside of the cantilever to prevent unwanted ligand adsorption, and this process requires tedious optimization. Here, we show a way to immobilize membrane receptors on nanomechanical cantilevers so that they can function without passivating the underlying surface. Using equilibrium theory, we quantitatively describe the mechanical responses of vancomycin, human immunodeficiency virus type 1 antigens and coagulation factor VIII captured on the cantilever in the presence of competing stresses from the top and bottom cantilever surfaces. We show that the area per receptor molecule on the cantilever surface influences ligand-receptor binding and plays an important role on stress. Our results offer a new way to sense biomolecules and will aid in the creation of ultrasensitive biosensors