A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteria : research data underlying the article

Jablonska, Maša (avtor)

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.

• Jezik: Angleški jezik
• Ključne besede: cyanotoxins, water management, early warning, risk assessment, qPCR, PCR, LC-MS/MS, ana gene cluster, anaC gene, DNA sequencing
• Tipologija: 2.20 - Zaključena znanstvena zbirka raziskovalnih podatkov
• Geografsko pokritje: Ljubljana, Slovenia
• Časovno pokritje: From/Od 2021-12-08 to/do 2024-04-08
• Organizacija: BF - Biotehniška fakulteta
• Leto izida: 2026
• PID: 20.500.12556/RUL-182456
• Metode zbir. podat.: Poskus: Laboratorij; Zbiranje; Sinteza
• Datum objave v RUL: 18.05.2026

Licence

• Licenca: CC BY 4.0, Creative Commons Priznanje avtorstva 4.0 Mednarodna
• Povezava: http://creativecommons.org/licenses/by/4.0/deed.sl
• Opis: To je standardna licenca Creative Commons, ki daje uporabnikom največ možnosti za nadaljnjo uporabo dela, pri čemer morajo navesti avtorja.

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Nov kvantitativni PCR test za zaznavanje cianobakterij s potencialom za proizvodnjo anatoksinov : raziskovalni podatki, obravnavani v članku

Izvleček

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.

• Ključne besede: cianotoksini, upravljanje voda, zgodnje zaznavanje, ocena tveganja, qPCR, PCR, LC-MS/MS, genski grozd ana, gen anaC, sekvenciranje DNA