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Strohmeier O.,Hsg Imit Institute For Mikro Und Informationstechnik | Strohmeier O.,Albert Ludwigs University of Freiburg | Keil S.,Hsg Imit Institute For Mikro Und Informationstechnik | Kanat B.,Hsg Imit Institute For Mikro Und Informationstechnik | And 9 more authors.
RSC Advances | Year: 2015

We present total nucleic acid extraction from whole blood, Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, and Rift Valley fever RNA virus on a low-cost, centrifugal microfluidic LabDisk cartridge processed in a light-weight (<2 kg) and portable processing device. Compared to earlier work on disk based centrifugal microfluidics, this includes the following advances: combined lysis and nucleic acid purification on one cartridge and handling of sample volumes as large as 200 μL. The presented system has been validated for logarithmic dilutions of aforementioned bacteria and viruses from various sample matrices including blood plasma and culture media and extraction of human DNA from whole blood. Recovered DNA and RNA concentrations in the eluate were characterized by quantitative PCR to: 58.2-98.5%, 45.3-102.1% and 29.5-34.2% versus a manual reference for Bacillus subtilis, Escherichia coli and Rift Valley fever virus, respectively. For extraction of human DNA from whole blood, similar results for on-disk ((10.1 ± 7.6) × 104 DNA copies) and manual reference extraction ((10.2 ± 6.3) × 104 DNA copies) could be achieved. Eluates from on-disk extraction show slightly increased ethanol concentrations of 4.1 ± 0.3% to 5.5 ± 0.2% compared to a manual reference (2.0 ± 0.5% to 3.6 ± 0.6%). The complete process chain for sample preparation is automatically performed within ∼30 minutes, including ∼15 minutes lysis time. It is amenable to concatenation with downstream modules for multiplex nucleic acid amplification as recently demonstrated for panel testing of various pathogens at the point of care. This journal is © The Royal Society of Chemistry 2015.

Kunze A.,TU Munich | Dilcher M.,University of Gottingen | Abd El Wahed A.,University of Gottingen | Hufert F.,Institute of Microbiology and Virology | And 2 more authors.
Analytical Chemistry | Year: 2016

This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39°C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 103 GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches. © 2015 American Chemical Society.

Abd El Wahed A.,German Primate Center | Abd El Wahed A.,Mansoura University | Weidmann M.,University of Stirling | Hufert F.T.,Institute of Microbiology and Virology
Journal of Clinical Virology | Year: 2015

Background: In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. Objectives: The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. Study design: A suitcase laboratory "Diagnostics-in-a-Suitcase" (56 cm × 45.5 cm × 26.5 cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. Results: The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42 °C). The results were obtained within 2-7 min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. Conclusions: The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection. © 2015 The Authors.

Kamzolova S.V.,Russian Academy of Sciences | Vinokurova N.G.,Russian Academy of Sciences | Shemshura O.N.,Institute of Microbiology and Virology | Bekmakhanova N.E.,Institute of Microbiology and Virology | And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2014

The production of α-ketoglutaric acid by yeast Yarrowia lipolytica VKMY-2412 from ethanol and its subsequent chemical conversion to succinic acid (SA) were investigated. A highly effective and environmentally friendly process of α-ketoglutaric acid production was developed using a special pH-controlling strategy, in which the titration of the culture broth with KOH in the acid-formation phase was minimal, that allowed accumulation of only low amounts of inorganic wastes in the course of SA recovery. The culture broth filtrate containing α-ketoglutaric acid (88.7 g l-1) was directly employed for SA production; the amount of SA produced comprised 71.7 g l-1 with the yield of 70 % from ethanol consumed. SA was isolated from the culture broth filtrate in a crystalline form with the purity of 100 %. The yield of isolated SA was as high as 72 % of its amount in the culture broth filtrate. The antimicrobial and nematocidic effects of SA of microbial origin on pathogenic organisms that cause human and plant diseases were revealed for the first time. © 2014 Springer-Verlag.

Dmitrieva L.,University of Leeds | Kondakov A.A.,Russian Academy of Sciences | Oleynikov E.,Russian Academy of Sciences | Kydyrmanov A.,Institute of Microbiology and Virology | And 5 more authors.
PLoS ONE | Year: 2013

The Caspian seal (Pusa caspica) has declined by more than 90% since 1900 and is listed as endangered by IUCN. We made the first quantitative assessment of Caspian seal by-catch mortality in fisheries in the north Caspian Sea by conducting semi-structured interviews in fishing communities along the coasts of Russia (Kalmykia, Dagestan), Kazakhstan and Turkmenistan. We recorded a documented minimum by-catch of 1,215 seals in the survey sample, for the 2008-2009 fishing season, 93% of which occurred in illegal sturgeon fisheries. Due to the illegal nature of the fishery, accurately quantifying total fishing effort is problematic and the survey sample could reflect less than 10% of poaching activity in the north Caspian Sea. Therefore total annual by-catch may be significantly greater than the minimum documented by the survey. The presence of high by-catch rates was supported independently by evidence of net entanglement from seal carcasses, during a mass stranding on the Kazakh coast in May 2009, where 30 of 312 carcasses were entangled in large mesh sturgeon net remnants. The documented minimum by-catch may account for 5 to 19% of annual pup production. Sturgeon poaching therefore not only represents a serious threat to Caspian sturgeon populations, but may also be having broader impacts on the Caspian Sea ecosystem by contributing to a decline in one of the ecosystem's key predators. This study demonstrates the utility of interview-based approaches in providing rapid assessments of by-catch in illegal small-scale fisheries, which are not amenable to study by other methods. © 2013 Dmitrieva et al.

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