Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV

Cargo-specific effects of hypoxia on clathrin-mediated trafficking

Clathrin-associated trafficking is a major mechanism for intracellular communication, as well as for cells to communicate with the extracellular environment. A decreased oxygen availability termed hypoxia has been described to influence this mechanism in the past. Mostly biochemical studies were applied in these analyses, which miss spatiotemporal information. We have applied live cell microscopy and a newly developed analysis script in combination with a GFP-tagged clathrin-expressing cell line to obtain insight into the dynamics of the effect of hypoxia. Number, mobility and directionality of clathrin-coated vesicles were analysed in non-stimulated cells as well as after stimulation with epidermal growth factor (EGF) or transferrin in normoxic and hypoxic conditions. These data reveal cargo-specific effects, which would not be observable with biochemical methods or with fixed cells and add to the understanding of cell physiology in hypoxia. The stimulus-dependent consequences were also reflected in the final cellular output, i.e. decreased EGF signaling and in contrast increased iron uptake in hypoxia.</p

Ablutions et bains rituels chez les Seereer Siin du Sénégal

Note portant sur l’auteur Note portant sur l’auteur Note portant sur l’auteur Les pratiques de bains rituels et de lavages sont largement répandues dans la médecine traditionnelle Seereer. Elles révèlent différents modes de transmission des connaissances médicales. Le savoir populaire Les femmes ont une connaissance des plantes utilisées pour les lavages thérapeutiques pour les maux les plus répandus. Elles utilisent les plantes qui se trouvent à leur portée, près de la maison et des champs. ..

Du savoir commun à la connaissance de la nuit chez les Seereer Siin du Sénégal

Note portant sur l'auteur Note portant sur l'auteur Note portant sur l'auteur Le savoir populaire Le savoir populaire est accessible à tous. Il est souvent exempt de tout support rituel, il n'utilise les plantes et ne manipule le verbe que dans le but de soulager un mal symptomatique. Il se prodigue la plupart du temps dans le champ restreint de la famille, d'amis ou de voisins. Il est véhiculé gratuitement. Les femmes restent les soignantes privilégiées dans le domaine des affections courant..

Le massage, une forme de traitement dans la médecine traditionnelle chez les Seereer Siin du Sénégal

Note portant sur l'auteur Note portant sur l'auteur Note portant sur l'auteur En observant les pratiques du guérisseur Geidj Faye, nous avons constaté l'importance et le rôle symbolique des massages dans la médecine traditionnelle Seereer. Ils constituent un bon exemple de la transmission des connaissances car ils relèvent d'un savoir secret qui est perpétué dans la famille, ce guérisseur étant le quatrième détenteur dans la lignée paternelle. En même temps, les explications du mode de foncti..

Cargo-specific effects of hypoxia on clathrin-mediated trafficking

Clathrin-associated trafficking is a major mechanism for intracellular communication, as well as for cells to communicate with the extracellular environment. A decreased oxygen availability termed hypoxia has been described to influence this mechanism in the past. Mostly biochemical studies were applied in these analyses, which miss spatiotemporal information. We have applied live cell microscopy and a newly developed analysis script in combination with a GFP-tagged clathrin-expressing cell line to obtain insight into the dynamics of the effect of hypoxia. Number, mobility and directionality of clathrin-coated vesicles were analysed in non-stimulated cells as well as after stimulation with epidermal growth factor (EGF) or transferrin in normoxic and hypoxic conditions. These data reveal cargo-specific effects, which would not be observable with biochemical methods or with fixed cells and add to the understanding of cell physiology in hypoxia. The stimulus-dependent consequences were also reflected in the final cellular output, i.e. decreased EGF signaling and in contrast increased iron uptake in hypoxia.</p

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&deg;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

Sequence of primers and probes for the construction of molecular standards, real-time RT-PCR and RT-RPA assays.

<p>Bp, base pair; DENV1-3-STD-UP/DP are for DENV1-3 RT-PCR; DENV4-STD-UP/DP are for DENV4 RT-PCR; DENV-PCR-FP/RP/P, real-time RT-PCR primers and Taqman probe (FAM/TAMRA); DENV1-3-RPA-FP, forward and reverse primers for DENV1-3 RT-RPA; DENV4-RPA-FP, forward and reverse primers for DENV4 RT-RPA; DENV-RPA-P, exo-probe; BHQ1-dT: thymidine nucleotide carrying Blackhole quencher1, THF: tetrahydrofuran spacer, FAM-dT: thymidine nucleotide carrying Fluorescein. N/A, non applicable.</p><p>Sequence of primers and probes for the construction of molecular standards, real-time RT-PCR and RT-RPA assays.</p

List of viral RNA tested.

<p>DENV1-3 RT-RPA assay detected DENV1-3 but not other viral RNA. DENV4 RT-RPA identified DENV2 and DENV4.</p><p>+, positive;-, negative.</p><p>List of viral RNA tested.</p