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
BACKGROUND: Detection of nucleic acids of Rift Valley fever virus (RVFV) has been shown to be useful in field diagnostics. OBJECTIVES: To develop an isothermal 'recombinase polymerase amplification (RPA)' assay on an ESEquant tubescanner device. STUDY DESIGN: RPA was adapted for RNA amplification by first developing a two-step and then a one-step-RT-RPA protocol. Several RT enzymes were tested and the best sensitivity was achieved using Transcriptor (Roche). Finally an RT-RPA pellet containing a recombinant MuLV was tested in RVFV one-step-RT-RPA. RESULTS: The one-step-RT-RPA assay showed a sensitivity of 19 molecules detected as determined by probit analysis of eight runs using a RVFV S-segment based quantitative RNA standard and detected 20 different RVFV strains. The assays showed no cross detection of the human genome and several agents of a typical biothreat panel. It performed almost as good as the assay using glycerol buffer based Transcriptor albeit at a cost of 1-log(10) step in sensitivity. The presented combination of one-step-RT-RPA and portable fluorescence reading device could be a useful tool for field or point of care diagnostics.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Febre do Vale de Rift/diagnóstico , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/isolamento & purificação , Animais , Colorimetria/métodos , Humanos , RNA Viral/genética , Vírus da Febre do Vale do Rift/genética , Sensibilidade e Especificidade , Fatores de TempoRESUMO
Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.
