Development and application of a multiplex qPCR te

Authors: Felexce F Ngwa Chandra A Madramootoo Suha Jabaji

Publish Date: 2013/11/23

Volume: 26, Issue: 4, Pages: 1675-1687

PDF Link

Abstract

The emergence and persistence of complex blooms comprising multiple toxigenic cyanobacteria genera pose significant challenges for water quality management worldwide The cooccurrence of morphologically indistinguishable toxic and nontoxic strains makes monitoring and control of these noxious organisms particularly challenging Conventional monitoring approaches are not only incapable of discriminating toxic from nontoxic strains but also have proven to be less sensitive and specific In this study a multiplex quantitative realtime 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 3amino9methoxy268trimethyl10phenyldeca46dienoic 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 099 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 microcystinproducing 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 approachesThe 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

Keywords: