TY - CONF A1 - Martin-Sanchez, Pedro Maria T1 - Quantitative PCR Assays for Monitoring Microorganisms Associated with Biodeterioration Cases T2 - XXV Spanish Microbiology Society Congress CY - Logrono, Spain DA - 2015-07-06 PY - 2015 AN - OPUS4-34255 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Martin-Sanchez, Pedro Maria T1 - Detection and quantification methods for material-colonizing microorganisms by pPCR: from rock paintings in Lascaux to photovoltaic panels T2 - DECHEMA /GfKORR-Fachgruppensitzung "Mikrobielle Materialzerstörung und Materialschutz" CY - Frankfurt am Main, Deutschland DA - 2014-10-17 PY - 2014 AN - OPUS4-33172 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Bell, Jérémy A1 - Climent, Estela A1 - Gotor, Raúl A1 - Tobias, Charlie A1 - Martin-Sanchez, Pedro M. A1 - Rurack, Knut T1 - Dipstick coated with polystyrene-silica core-shell particles for the detection of microbiological fuel contamination N2 - Microbial contamination of fuels by fungi or bacteria poses risks such as corrosion and fuel system fouling, which can lead to critical problems in refineries and distribution systems and has a significant economic impact at every stage of the process. Many factors have been cited as being responsible for microbial growth, like the presence of water in the storage tanks. In fact, only 1 % water in a storage system is sufficient for the growth of microorganisms like bacteria or yeasts, as well as for the development of fungal biomass at the oil/water interface. This work presents a rapid test for the accurate determination of genomic DNA from aqueous fuel extracts. The detection is based on the use of polystyrene-mesoporous silica core-shell particles onto which modified fluorescent molecular beacons are covalently grafted. These beacons contain in the hairpin loop a target sequence highly conserved in all bacteria, corresponding to a fragment of the 16S ribosomal RNA subunit. The designed single-stranded molecular beacon contained fluorescein as an internal indicator and a quencher in its proximity when not hybridized. Upon hybridization in presence of the target sequence, the indicator and the quencher are spatially separated, resulting in fluorescence enhancement. To perform the assay the developed particles were deposited on different glass fibre strips to obtain a portable and sensitive rapid test. The assays showed that the presence of genomic DNA extracts from bacteria down to 50–70 μg L–1 induced a fluorescence response. The optical read-out was adapted for on-site monitoring by fitting a 3D-printed case to a conventional smartphone, taking advantages of the sensitivity of the CMOS detector. Such embedded assembly enabled the detection of genomic DNA in aqueous extracts down to the mg L–1 range and represents an interesting step toward on-site monitoring of fuel contamination. T2 - IMA 2023 CY - Chania, Greece DA - 18.09.2023 KW - Teststreifen KW - Test strip KW - Microbial KW - Mikrobiell KW - Smartphone KW - Particles KW - Partikeln PY - 2023 AN - OPUS4-58526 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Martin-Sanchez, Pedro Maria T1 - A novel qPCR protocol to the specific detection and quantification of Hormoconis resinae in fuel systems N2 - A novel qPCR protocol to the specific detection and quantification of Hormoconis resinae in fuel systems P.M. Martin-Sanchez1 , J. Toepel1, H.J. Kunte1, A.A. Gorbushina1, 2 1BAM Federal Institute for Materials Research and Testing, Department 4 Materials & Environment, Berlin, Germany 2 Free University of Berlin, Department of Earth Sciences & Biology, Chemistry & Pharmacy, Berlin, Germany Introduction. Microbial contamination of fuels is a phenomenon widely reported in the literature causing dramatic problems in fuel systems such as blockage of pipelines and filters, and corrosion of their metal components. A variety of microorganisms, fungi, yeasts and bacteria, can contaminate the distillate fuels. Between them, the filamentous fungus Hormoconis resinae, previously described as Cladosporium resinae and also known as "kerosene fungus" or "diesel bug", has traditionally been considered the main responsible of such troubles due to its great capacity to produce biomass and biofilms. Aim. The overarching goal of this study was to develop a novel real-time quantitative PCR (qPCR) method to detect and quantify the fungus H. resinae in samples from fuel systems. Materials and methods. Four specific primer sets targeting on two molecular markers, RNA polymerase II second largest subunit (RPB2) and internal transcribed spacers of rDNA (ITS), were designed using Primer Blast. The specificity of primer sets was evaluated by both conventional PCR and qPCR, analyzing the DNA extracts from H. resinae strains and other fungi and yeasts previously described in fuels as well as four taxonomically closest related species. The efficiency and sensitivity of these protocols were assessed by constructing the corresponding standard curves for each primer sets. Results. Two primer sets, Hr556F/Hr814R (RPB2) and Hr101F/Hr408R (ITS), were selected according to their remarkable specificity. Their standard curves showed a good correlation coefficient and efficiency. Additionally, the selected primer pairs showed a high sensitivity detecting a tiny amount of H. resinae DNA, 10 pg for RPB2 primers and 0.1 pg for ITS primers. Conclusions. The established qPCR protocol is a reliable and useful tool to the specific detection and quantification of H. resinae. Its subsequent implementation to analyze samples of fuels or biofilms covering fuel systems will allow the early detection of fungal outbreaks in order to apply the appropriated control procedures. Moreover, these analyses will determine the current incidence of this species in the cases of biological fuel contamination. Keywords. Hormoconis resinae, real-time qPCR, diesel bug, kerosene fungus T2 - XVI International Biodeterioration and Biodegradation Symposium; International Biodeterioration & Biodegradation Society CY - Lodz, Poland DA - 2014-09-03 PY - 2014 AN - OPUS4-32165 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -