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Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake

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Abstract

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide. The co-occurrence of morphologically indistinguishable toxic and non-toxic strains makes monitoring and control of these noxious organisms particularly challenging. Conventional monitoring approaches are not only incapable of discriminating toxic from non-toxic strains but also have proven to be less sensitive and specific. In this study, a multiplex quantitative real-time polymerase chain reaction (qPCR) approach was developed and tested for its sensitivity and specificity at detecting, differentiating and estimating potentially toxic Anabaena, Microcystis and Planktothrix genotype compositions in environmental samples. The oligonucleotide primers and probes utilized were designed to target portions of the microcystin synthetase (mcy) E gene that encode synthesis of the unique 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (ADDA) moiety of microcystins in the three target genera. Laboratory evaluation showed the developed assay to be highly sensitive and specific at detecting and quantifying targeted genera. Indeed, the assay standards for the Anabaena, Microcystis and Planktothrix reactions attained efficiencies above 90 %, with coefficients of determination consistently above 0.99. Analysis of water samples from Missisquoi Bay, Quebec, Canada, resulted in successful detection and quantification of target toxigenic cyanobacteria even when cell numbers were below the detection limit for the conventional microscopy methods. Furthermore, toxigenic Microcystis spp. were found to be the main putative microcystin-producing cyanobacteria in the study lake. The qPCR technique developed in this study therefore offers simultaneous detection, differentiation and quantification of multiple toxigenic cyanobacteria that otherwise cannot be accomplished by current monitoring approaches.

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Acknowledgements

The authors thank T. Coplay and R. Chamoun for helpful discussions on assay design and optimization. This research was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grants to C. Madramootoo and S. Jabaji and the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) grant to C. Madramootoo. Hedy Kling of Algal Taxonomy and Ecology is gratefully acknowledged for assistance with phytoplankton taxonomy and enumeration.

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  1. Department of Bioresource Engineering, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste. Anne de Bellevue, Québec, H9X 3V9, Canada

    Felexce F. Ngwa & Chandra A. Madramootoo

  2. Department of Plant Science, McGill University, Macdonald Campus, 21111 Lakeshore Road, Ste. Anne de Bellevue, Québec, H9X 3V9, Canada

    Suha Jabaji

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  1. Felexce F. Ngwa
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Correspondence to Felexce F. Ngwa.

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Ngwa, F.F., Madramootoo, C.A. & Jabaji, S. Development and application of a multiplex qPCR technique to detect multiple microcystin-producing cyanobacterial genera in a Canadian freshwater lake. J Appl Phycol 26, 1675–1687 (2014). https://doi.org/10.1007/s10811-013-0199-9

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