RESUMO
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.
Assuntos
Bactérias/isolamento & purificação , Armas Biológicas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Vírus/isolamento & purificação , Bactérias/genética , Humanos , Ácidos Nucleicos/genética , Ácidos Nucleicos/isolamento & purificação , Plasma/microbiologia , Plasma/virologia , Temperatura , Fatores de Tempo , Vírus/genéticaRESUMO
With a view to developing a rapid pathogen detection system utilizing isothermal nucleic acid amplification, the necessary micro-mixing step is innovatively implemented on a chip. Passive laminar flow mixing of two 6.5 µl batches differing in viscosity is performed within a microfluidic chamber. This is achieved with a novel chip space-saving phaseguide design which allows, for the first time, the complete integration of a passive mixing structure into a target chamber. Sequential filling of batches prior to mixing is demonstrated. Simulation predicts a reduction of diffusive mixing time from hours down to one minute. A simple and low-cost fabrication method is used which combines dry film resist technology and direct wafer bonding. Finally, an isothermal nucleic acid detection assay is successfully implemented where fluorescence results are measured directly from the chip after a one minute mixing sequence. In combination with our previous work, this opens up the way towards a fully integrated pathogen detection system in a lab-on-a-chip format.
Assuntos
Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/análise , Temperatura , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , ViscosidadeRESUMO
Macro- to micro-volume concentration of viable bacteria is performed in a microfluidic chip. The enrichment principle is based on free flow electrophoresis and is demonstrated for Gram positive bacteria. Bacteria from a suspension flow are trapped on a gel interface that separates the trapping location from integrated actuation electrodes in order to enable non-destructive trapping. The microfluidic chip contains integrated electrolytic gas expulsion structures and phaseguides for gel and liquid handling. Trapping efficiency is systematically optimized to reach 25 times the initial concentration from a theoretical maximum of 30. Finally, enrichment from analytically relevant concentrations down to 3 × 10(2) colony forming units per millilitre is demonstrated with a trapping efficiency of 80% which represents the most important parameter in enrichment.