1. Proficiency testing and cross-laboratory method comparison to support standardisation of diatom DNA metabarcoding for freshwater biomonitoringValentin Vasselon, Sinziana F. Rivera, Éva Ács, Tina Eleršek, Maša Jablonska, 2025, izvirni znanstveni članek Povzetek: DNA metabarcoding of benthic diatoms has been successfully applied for biomonitoring at the national scale and can now be considered technically ready for routine application. However, protocols and methods still vary between and within countries, limiting their transferability and the comparability of results. In order to overcome this, routine use of DNA metabarcoding for diatom biomonitoring requires knowledge of the sources of variability introduced by the different steps of the procedure. Here, we examine how elements of routine procedures contribute to variability between European laboratories. A set of four experiments were performed focusing on DNA extraction and PCR amplification steps to evaluate their reproducibility between different laboratories and the variability introduced by different protocols currently applied by the scientific community. Under the guidance of a reference laboratory, 17 participants from 14 countries performed DNA extraction and PCR amplification in parallel, using the same fixed protocol and their own choice of protocol. Experiments were performed by each participant on a set of standardised DNA and biofilm samples (river, lake and mock community) to investigate potential systematic and random errors. Our results revealed the successful transferability of a protocol amongst labs and a highly similar and consistent ecological assessment outcome obtained regardless of the protocols used by each participant. We propose an “all for one but prove them all” strategy, suggesting that distinct protocols can be used within the scientific community, as long as their consistency is be proven by following minimum standard requirements.
Ključne besede: cross-laboratory experiment, DNA-based approach, ecological status assessment, intercalibration, standardisation, environmental science, biomonitoring, DNA metabarcoding, benthic diatoms
Objavljeno v DiRROS: 17.01.2025; Ogledov: 317; Prenosov: 210
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2. A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteriaMaša Jablonska, Tina Eleršek, 2025, izvirni znanstveni članek Povzetek: Anatoxins (ATX) are globally occurring toxins produced by some species of cyanobacteria in aquatic habitats. They can cause acute poisoning in animals, leading to muscle paralysis and respiratory failure, and might also pose a long-term health risk to humans. Thanks to advances in molecular methods and genomic knowledge, it is now possible to rapidly detect and quantify the genes associated with cyanotoxin production for most major groups of cyanotoxins except ATX. The aim of this study was to develop and validate a new quantitative PCR (qPCR) assay for general detection of all potential ATX producers in the environment. After specificity testing in silico and in vitro with 16 cyanobacterial strains (endpoint PCR, amplicon sequencing and qPCR), two assays targeting the anaC gene were thoroughly validated for linearity, amplification efficiency, sensitivity, dynamic range, inter-assay and intra-assay variability, and the influence of background DNA. The assays were then applied to 144 environmental samples of plankton and biofilm from lakes and rivers whose ATX content had previously been measured by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Amplification efficiency of the two designed assays was between 94% and 103%, and the limits of quantification and detection were up to, but mostly below, 322 and 32 cells/mL, respectively. Both assays showed better or equal specificity in cyanobacterial cultures than currently available PCR assays and were able to predict the presence of ATX detected by LC-MS/MS in most environmental samples (83 % in plankton and 52–62% in biofilm). A higher number of discrepancies between qPCR and LC-MS/MS results in biofilm than in plankton samples indicates limited knowledge and sparse genomic data on benthic cyanobacteria. These assays are the first published general qPCR assays targeting all ATX producers and could provide water managers with a rapid and cost-effective risk assessment to better protect human and animal health.
Ključne besede: anatoxins (ATX), cyanobacteria, qPCR assay, anaC gene, LC-MS/MS, environmental microbiology, environmental science, toxicology, molecular biology
Objavljeno v DiRROS: 17.12.2024; Ogledov: 451; Prenosov: 237
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3. Molecular screening for cyanobacteria and their cyanotoxin potential in diverse habitatsMaša Jablonska, Tina Eleršek, Polona Kogovšek, Sara Skok, Andreea Oarga-Mulec, Janez Mulec, 2024, izvirni znanstveni članek Povzetek: Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer—PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins—mcyE, saxitoxins—sxtA, cylindrospermopsins—cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments.
Ključne besede: extreme environments, cylindrospermopsin, sulphidic springs, caves, qPCR, PC-IGS
Objavljeno v DiRROS: 05.08.2024; Ogledov: 693; Prenosov: 412
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4. Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screeningMaša Jablonska, Leonardo Cerasino, Adriano Boscaini, Camilla Capelli, Claudia Greco, Aleksandra Krivograd-Klemenčič, Ute Mischke, Nico Salmaso, Rainer Kurmayer, 2024, izvirni znanstveni članek Povzetek: The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study.
Ključne besede: cyanotoxins, planktic and benthic habitats, water quality monitoring, early warning, toxigenic cyanobacteria, European Alps
Objavljeno v DiRROS: 03.06.2024; Ogledov: 979; Prenosov: 548
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