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A new quantitative PCR assay for the detection of hepatotoxigenic cyanobacteria
J Al-Tebrineh1, M M Gehringer, R Akcaalan
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia.
Abstract:
Toxin-producing cyanobacteria are a worldwide threat to both human and animal health. The hepatotoxins microcystin and nodularin are the most commonly occurring toxins produced by bloom-forming cyanobacteria. They are cyclic peptides that are synthesized nonribosomally by a multienzyme complexes encoded within the microcystin (mcyS) and nodularin (ndaS) synthetase gene clusters. Early detection of potentially toxic blooms would allow for pre-emptive action to reduce consumer exposure to cyanotoxins. We have developed a quantitative PCR (qPCR) assay based on SYBR-green chemistry for the detection of potentially hepatotoxic cyanobacteria spanning all known microcystin and nodularin producing taxa using primers specifically targeting mcyE and ndaF. The qPCR assay was validated against previously analyzed cyanobacterial bloom samples. Whole cell qPCR using cultured M. aeruginosa PCC7806 and non-toxic M. aeruginosa UTEX2386 had a sensitivity of 1000 cells ml⁻¹. In summary, we have developed a robust and sensitive molecular method for the detection and quantification of hepatotoxigenic cyanobacteria in bloom samples. This technology offers several advantages over traditional and contemporary testing protocols currently used to assess water quality.
Insights
A new quantitative PCR (qPCR) assay detects microcystin and nodularin producing cyanobacteria. This rapid molecular method enhances early warning systems for toxic algal blooms, protecting human and animal health.
Area of Science:
- Environmental Microbiology
- Molecular Biology
- Ecotoxicology
Background:
- Cyanobacteria blooms produce potent hepatotoxins, microcystins and nodularins, posing global health risks.
- These cyclic peptide toxins are synthesized by large enzyme complexes encoded by specific gene clusters (mcyS and ndaS).
- Timely detection of toxic blooms is crucial for preventing human and animal exposure to cyanotoxins.
Purpose of the Study:
- To develop a sensitive and robust molecular assay for detecting and quantifying hepatotoxic cyanobacteria.
- To target conserved genes (mcyE and ndaF) common to microcystin and nodularin producers.
- To provide a reliable tool for early warning of potentially harmful cyanobacterial blooms.
Main Methods:
- Development of a quantitative PCR (qPCR) assay utilizing SYBR-green chemistry.
- Design of specific primers targeting the mcyE and ndaF genes.
- Validation of the qPCR assay using cultured cyanobacteria and previously analyzed bloom samples.
Main Results:
- The qPCR assay successfully detected both microcystin and nodularin producing cyanobacteria.
- Whole cell qPCR demonstrated a sensitivity of 1000 cells/mL for cultured M. aeruginosa.
- The assay proved robust and sensitive when validated against environmental bloom samples.
Conclusions:
- A novel, sensitive, and robust qPCR method for detecting hepatotoxigenic cyanobacteria has been established.
- This molecular technology offers significant advantages over traditional water quality assessment methods.
- The assay facilitates early detection and quantification of toxic cyanobacteria, aiding in risk management.
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