A new quantitative PCR assay for detection of potentially anatoxin-producing cyanobacteria

Jablonska, Maša (author), Eleršek, Tina (author)

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.

• Language: English
• Keywords: anatoxins, ATX, cyanobacteria, qPCR assay, anaC gene, LC-MS/MS, environmental microbiology, environmental science, toxicology, molecular biology, cyanotoxins, water management, early warning, risk assessment, ana gene cluster
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: BF - Biotechnical Faculty
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 12 str.
• Numbering: Vol. 142, art. 102785
• PID: 20.500.12556/RUL-169762
• UDC: 579
• ISSN on article: 1878-1470
• DOI: 10.1016/j.hal.2024.102785
• COBISS.SI-ID: 219478275
• Publication date in RUL: 09.06.2025

Record is a part of a journal

• Title: Harmful algae
• Publisher: Elsevier
• ISSN: 1878-1470
• COBISS.SI-ID: 175350787

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: anatoksini, ATX, cianobakterije, qPCR test, gen anaC, okoljska mikrobiologija, znanosti o okolju, toksikologija, molekularna biologija