Development of a panel of recombinase polymerase amplification assays for detection of biothreat agents.

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis, Yersinia pestis, Bacillus anthracis, variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

Point-of-Care System for HTLV-1 Proviral Load Quantification by Digital Mediator Displacement LAMP.

This paper presents a universal point-of-care system for fully automated quantification of human T-cell lymphotropic virus type 1 (HTLV-1) proviral load, including genomic RNA, based on digital reverse RNA transcription and c-DNA amplification by MD LAMP (mediator displacement loop-mediated isothermal amplification). A disposable microfluidic LabDisk with pre-stored reagents performs automated nucleic acid extraction, reaction setup, emulsification, reverse transcription, digital DNA amplification, and quantitative fluorogenic endpoint detection with universal reporter molecules. Automated nucleic acid extraction from a suspension of HTLV-1-infected CD4+ T-lymphocytes (MT-2 cells) yielded 8 ± 7 viral nucleic acid copies per MT-2 cell, very similar to the manual reference extraction (7 ± 2 nucleic acid copies). Fully automated sample processing from whole blood spiked with MT-2 cells showed a comparable result of 7 ± 3 copies per MT-2 cell after a run time of two hours and 10 min.

Chair/bedside diagnosis of oral and respiratory tract infections, and identification of antibiotic resistances for personalised monitoring and treatment.

Global healthcare systems are struggling with the enormous burden associated with infectious diseases, as well as the incessant rise of antimicrobial resistance. In order to adequately address these issues, there is an urgent need for rapid and accurate infectious disease diagnostics. The H2020 project DIAGORAS aims at diagnosing oral and respiratory tract infections using a fully integrated, automated and user-friendly platform for physicians' offices, schools, elderly care units, community settings, etc. Oral diseases (periodontitis, dental caries) will be detected via multiplexed, quantitative analysis of salivary markers (bacterial DNA and host response proteins) for early prevention and personalised monitoring. Respiratory Tract Infections will be diagnosed by means of DNA/RNA differentiation so as to identify their bacterial or viral nature. Together with antibiotic resistance screening on the same platform, a more efficient treatment management is expected at the point-of-care. At the heart of DIAGORAS lies a centrifugal microfluidic platform (LabDisk and associated processing device) integrating all components and assays for a fully automated analysis. The project involves an interface with a clinical algorithm for the comprehensive presentation of results to end-users, thereby increasing the platform's clinical utility. DIAGORAS' performance will be validated at clinical settings and compared with gold standards.

Rapid molecular assays for the detection of yellow fever virus in low-resource settings.

BACKGROUND: Yellow fever (YF) is an acute viral hemorrhagic disease transmitted by Aedes mosquitoes. The causative agent, the yellow fever virus (YFV), is found in tropical and subtropical areas of South America and Africa. Although a vaccine is available since the 1930s, YF still causes thousands of deaths and several outbreaks have recently occurred in Africa. Therefore, rapid and reliable diagnostic methods easy to perform in low-resources settings could have a major impact on early detection of outbreaks and implementation of appropriate response strategies such as vaccination and/or vector control. METHODOLOGY: The aim of this study was to develop a YFV nucleic acid detection method applicable in outbreak investigations and surveillance studies in low-resource and field settings. The method should be simple, robust, rapid and reliable. Therefore, we adopted an isothermal approach and developed a recombinase polymerase amplification (RPA) assay which can be performed with a small portable instrument and easy-to-use lyophilized reagents. The assay was developed in three different formats (real-time with or without microfluidic semi-automated system and lateral-flow assay) to evaluate their application for different purposes. Analytical specificity and sensitivity were evaluated with a wide panel of viruses and serial dilutions of YFV RNA. Mosquito pools and spiked human plasma samples were also tested for assay validation. Finally, real-time RPA in portable format was tested under field conditions in Senegal. CONCLUSION/SIGNIFICANCE: The assay was able to detect 20 different YFV strains and demonstrated no cross-reactions with closely related viruses. The RPA assay proved to be a robust, portable method with a low detection limit (<21 genome equivalent copies per reaction) and rapid processing time (<20 min). Results from real-time RPA field testing were comparable to results obtained in the laboratory, thus confirming our method is suitable for YFV detection in low-resource settings.

Centrifugal gas-phase transition magnetophoresis (GTM)--a generic method for automation of magnetic bead based assays on the centrifugal microfluidic platform and application to DNA purification.

Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge "LabDisk". The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

Carbon electrodes for direct electron transfer type laccase cathodes investigated by current density-cathode potential behavior.

Direct electron transfer from carbon electrodes to adsorbed laccase (EC 1.10.3.2) from Trametes versicolor is widely used to enable mediatorless enzymatic biofuel cell cathodes. However, data published so far are poorly comparable in terms of oxygen reduction performance. We thus present a comparative characterization of carbon-based electrode materials as cathode in half-cell configuration, employing adsorbed laccase as oxygen reduction catalyst. Open circuit potentials and performances were significantly increased by laccase adsorption, indicating the occurrence of direct electron transfer. At a potential of 0.5 V vs. SCE volume-normalized current densities of approximately 10, 37, 40, 70, and 77 µA cm(-3) were measured for cathodes nanotubes, carbon nanofibers and multi-walled carbon nanotubes, respectively. In addition, we could show that both, carbon nanotubes and porous carbon tubes exhibit dramatically lower current densities compared to graphite felt and carbon nanofibers when normalized to BET surface instead of electrode volume. Further work will be required to clarify whether this stems from material-dependent interaction of enzyme and electrode surface or constricted enzyme adsorption due to agglomeration of the nanotubes. In case of the latter, an improved dispersion of the nanotubes upon electrode fabrication may greatly enhance their performance.