20.500.12556/RUL-182456 A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteria research data underlying the article Nov kvantitativni PCR test za zaznavanje cianobakterij s potencialom za proizvodnjo anatoksinov raziskovalni podatki, obravnavani v članku 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. Anatoksini (ATX) so globalno razširjeni toksini, ki jih proizvajajo nekatere vrste cianobakterij v vodnih habitatih. Pri živalih lahko povzročijo akutno zastrupitev, kar vodi do mišične paralize in odpovedi dihanja, poleg tega pa lahko predstavljajo tudi dolgoročno tveganje za zdravje ljudi. Zahvaljujoč napredku molekularnih metod in poznavanja genomike je danes mogoče hitro zaznati in kvantificirati gene, povezane s proizvodnjo večine glavnih skupin cianotoksinov, razen ATX. Namen te raziskave je bil razviti in ovrednotiti nov kvantitativni test PCR (qPCR) za splošno zaznavanje vseh potencialnih proizvajalcev ATX v okolju. Po testiranju specifičnosti in silico in in vitro s 16 cianobakterijskimi sevi (klasičen PCR, sekvenciranje amplikonov in qPCR) smo za dva testa za zaznavanje gena anaC natančno ovrednotili linearnost, učinkovitost pomnoževanja, občutljivost, dinamično območje, variabilnost znotraj analize in med analizami ter vpliv netarčne DNA. Teste smo nato uporabili za analizo 144 okoljskih vzorcev planktona in bentosa iz jezer in rek, katerih vsebnost ATX je bila predhodno izmerjena s tekočinsko kromatografijo sklopljeno s tandemsko masno spektrometrijo (LC-MS/MS). Učinkovitost pomnoževanja oblikovanih testov je bila med 94 % in 103 %, meje kvantifikacije in zaznave pa so bile do 322 in 32 celic/mL. Oba testa sta pokazala boljšo ali enako specifičnost v cianobakterijskih kulturah kot trenutno dostopni PCR testi in sta lahko napovedala prisotnost ATX potrjeno z LC-MS/MS v večini okoljskih vzorcev (83 % v planktonu in 52–62 % v bentosu). Neskladja med rezultati qPCR in LC-MS/MS so bila pogostejša v vzorcih bentosa kot v vzorcih planktona, kar kaže na omejeno poznavanje in pomanjkljive genomske podatke o bentoških cianobakterijah. Ta testa sta prva objavljena testa qPCR za zaznavanje vseh proizvajalcev ATX in bi lahko upravljavcem vodnih teles omogočila hitro in cenovno ugodno oceno tveganja za boljšo zaščito zdravja ljudi in živali. cyanotoxins water management early warning risk assessment qPCR PCR LC-MS/MS ana gene cluster anaC gene DNA sequencing cianotoksini upravljanje voda zgodnje zaznavanje ocena tveganja qPCR PCR LC-MS/MS genski grozd ana gen anaC sekvenciranje DNA true false false Angleški jezik Slovenski jezik Ljubljana, Slovenia From/Od 2021-12-08 to/do 2024-04-08 Neznano 2026-05-12 12:26:22 2026-05-18 09:16:40 2026-05-19 04:36:14 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 README.txt README.txt README 1 95068F2AC9DC06EC521D99F5D725535D 841e7a17caf222f91e1f53ea19ab233efd52d869fb672580864c4866637ace88 febdd4d3-4def-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233740 PREBERI_ME.txt PREBERI_ME.txt PREBERI ME 1 DB9576F76AD34D09F19D878291C8F631 cbb437f5973d8a6a3ef3d24bb79cd2b8e863d1148133fe4f5ab740fafef26008 feab0fe3-4def-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233739 data.zip data.zip Data 1 9CE37AB816393E13E44DE8F5C8170ADC d2e78acebb22cbebda82ec45fb74dd178d1919b94dba75f4f63727a621e9966d fdc5a82a-4def-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233738 https://doi.org/10.1016/j.hal.2024.102785 Article 0 cd46a677-4dec-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=233732 Biotehniška fakulteta Poskus: Laboratorij Zbiranje Sinteza A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteria 0 0 0