Journal of Applied Phycology

, Volume 26, Issue 4, pp 1675–1687

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

  • Felexce F. Ngwa
  • Chandra A. Madramootoo
  • Suha Jabaji
Article

DOI: 10.1007/s10811-013-0199-9

Cite this article as:
Ngwa, F.F., Madramootoo, C.A. & Jabaji, S. J Appl Phycol (2014) 26: 1675. doi:10.1007/s10811-013-0199-9

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.

Keywords

Multiplex qPCRMicrocystin synthetaseMicrocystinWater qualityCyanobacterial blooms

Supplementary material

10811_2013_199_MOESM1_ESM.docx (23 kb)
ESM 1(DOCX 23 kb)

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Felexce F. Ngwa
    • 1
  • Chandra A. Madramootoo
    • 1
  • Suha Jabaji
    • 2
  1. 1.Department of Bioresource EngineeringMcGill UniversityQuébecCanada
  2. 2.Department of Plant ScienceMcGill UniversityQuébecCanada