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- ItemDNA and RNA extraction and quantitative real-time PCR-based assays for biogas biocenoses in an interlaboratory comparison(Basel : MDPI, 2016) Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, MichaelFive institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported.
- ItemIsolation of bacteria from artificial bronchoalveolar lavage fluid using density gradient centrifugation and their accessibility by Raman spectroscopy(Berlin [u.a.] : Springer, 2021) Wichmann, Christina; Rösch, Petra; Popp, JürgenRaman spectroscopy is an analytical method to identify medical samples of bacteria. Because Raman spectroscopy detects the biochemical properties of a cell, there are many factors that can influence and modify the Raman spectra of bacteria. One possible influence is a proper method for isolation of the bacteria. Medical samples in particular never occur in purified form, so a Raman-compatible isolation method is needed which does not affect the bacteria and thus the resulting spectra. In this study, we present a Raman-compatible method for isolation of bacteria from bronchoalveolar lavage (BAL) fluid using density gradient centrifugation. In addition to measuring the bacteria from a patient sample, the yield and the spectral influence of the isolation on the bacteria were investigated. Bacteria isolated from BAL fluid show additional peaks in comparison to pure culture bacteria, which can be attributed to components in the BAL sample. The isolation gradient itself has no effect on the spectra, and with a yield of 63% and 78%, the method is suitable for isolation of low concentrations of bacteria from a complex matrix. Graphical abstract.
- ItemDetection of ESKAPE bacterial pathogens at the point of care using isothermal DNA-based assays in a portable degas-actuated microfluidic diagnostic assay platform(Washington, DC [u.a.] : Soc., 2017) Renner, Lars D.; Zan, Jindong; Hu, Linda I.; Martinez, Manuel; Resto, Pedro J.; Siegel, Adam C.; Torres, Clint; Hall, Sara B.; Slezak, Tom R.; Nguyen, Tuan H.; Weibel, Douglas B.An estimated 1.5 billion microbial infections occur globally each year and result in ~4.6 million deaths. A technology gap associated with commercially available diagnostic tests in remote and underdeveloped regions prevents timely pathogen identification for effective antibiotic chemotherapies for infected patients. The result is a trial-and-error approach that is limited in effectiveness, increases risk for patients while contributing to antimicrobial drug resistance, and reduces the lifetime of antibiotics. This paper addresses this important diagnostic technology gap by describing a low-cost, portable, rapid, and easy-to-use microfluidic cartridgebased system for detecting the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacterial pathogens that are most commonly associated with antibiotic resistance. The point-of-care molecular diagnostic system consists of a vacuumdegassed microfluidic cartridge preloaded with lyophilized recombinase polymerase amplification (RPA) assays and a small portable battery-powered electronic incubator/ reader. The isothermal RPA assays detect the targeted ESKAPE pathogens with high sensitivity (e.g., a limit of detection of ~10 nucleic acid molecules) that is comparable to that of current PCR-based assays, and they offer advantages in power consumption, engineering, and robustness, which are three critical elements required for the point-of-care setting.
- ItemColiCoords: A Python package for the analysis of bacterial fluorescence microscopy data(San Francisco, California, US : PLOS, 2019) Smit, Jochem H.; Li, Yichen; Warszawik, Eliza M.; Herrmann, Andreas; Cordes, Thorben; Gilestro, Giorgio FSingle-molecule fluorescence microscopy studies of bacteria provide unique insights into the mechanisms of cellular processes and protein machineries in ways that are unrivalled by any other technique. With the cost of microscopes dropping and the availability of fully automated microscopes, the volume of microscopy data produced has increased tremendously. These developments have moved the bottleneck of throughput from image acquisition and sample preparation to data analysis. Furthermore, requirements for analysis procedures have become more stringent given the demand of various journals to make data and analysis procedures available. To address these issues we have developed a new data analysis package for analysis of fluorescence microscopy data from rod-like cells. Our software ColiCoords structures microscopy data at the single-cell level and implements a coordinate system describing each cell. This allows for the transformation of Cartesian coordinates from transmission light and fluorescence images and single-molecule localization microscopy (SMLM) data to cellular coordinates. Using this transformation, many cells can be combined to increase the statistical power of fluorescence microscopy datasets of any kind. ColiCoords is open source, implemented in the programming language Python, and is extensively documented. This allows for modifications for specific needs or to inspect and publish data analysis procedures. By providing a format that allows for easy sharing of code and associated data, we intend to promote open and reproducible research. The source code and documentation can be found via the project’s GitHub page.
- ItemNoise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications(Basel : MDPI, 2021) Jahn, Izabella J.; Grjasnow, Alexej; John, Henry; Weber, Karina; Popp, Jürgen; Hauswald, WalterRaman spectroscopy probes the biochemical composition of samples in a non-destructive, non-invasive and label-free fashion yielding specific information on a molecular level. Nevertheless, the Raman effect is very weak. The detection of all inelastically scattered photons with highest efficiency is therefore crucial as well as the identification of all noise sources present in the system. Here we provide a study for performance comparison and assessment of different spectrometers for confocal Raman spectroscopy in biosensor applications. A low-cost, home-built Raman spectrometer with a complementary metal-oxide-semiconductor (CMOS) camera, a middle price-class mini charge-coupled device (CCD) Raman spectrometer and a laboratory grade confocal Raman system with a deeply cooled CCD detector are compared. It is often overlooked that the sample itself is the most important “optical” component in a Raman spectrometer and its properties contribute most significantly to the signal-to-noise ratio. For this purpose, different representative samples: a crystalline silicon wafer, a polypropylene sample and E. coli bacteria were measured under similar conditions using the three confocal Raman spectrometers. We show that biosensor applications do not in every case profit from the most expensive equipment. Finally, a small Raman database of three different bacteria species is set up with the middle price-class mini CCD Raman spectrometer in order to demonstrate the potential of a compact setup for pathogen discrimination.
- ItemParticulate matter emissions during field application of poultry manure - The influence of moisture content and treatment(Amsterdam [u.a.] : Elsevier Science, 2021) Kabelitz, Tina; Biniasch, Oliver; Ammon, Christian; Nübel, Ulrich; Thiel, Nadine; Janke, David; Swaminathan, Senthilathiban; Funk, Roger; Münch, Steffen; Rösler, Uwe; Siller, Paul; Amon, Barbara; Aarnink, André J. A.; Amon, ThomasAlong with industry and transportation, agriculture is one of the main sources of primary particulate matter (PM) emissions worldwide. Bioaerosol formation and PM release during livestock manure field application and the associated threats to environmental and human health are rarely investigated. In the temperate climate zone, field fertilization with manure seasonally contributes to local PM air pollution regularly twice per year (spring and autumn). Measurements in a wind tunnel, in the field and computational fluid dynamics (CFD) simulations were performed to analyze PM aerosolization during poultry manure application and the influence of manure moisture content and treatment. A positive correlation between manure dry matter content (DM) and PM release was observed. Therefore, treatments strongly increasing the DM of poultry manure should be avoided. However, high manure DM led to reduced microbial abundance and, therefore, to a lower risk of environmental pathogen dispersion. Considering the findings of PM and microbial measurements, the optimal poultry manure DM range for field fertilization was identified as 50–70%. Maximum PM10 concentrations of approx. 10 mg per m3 of air were measured during the spreading of dried manure (DM 80%), a concentration that is classified as strongly harmful. The modeling of PM aerosolization processes indicated a low health risk beyond a distance of 400 m from the manure application source. The detailed knowledge about PM aerosolization during manure field application was improved with this study, enabling manure management optimization for lower PM aerosolization and pathogenic release into the environment.