Use of multiplex-tandem polymerase chain reaction in an operational environment to assess cyanotoxins and taste and odour metabolites of cyanobacterial blooms in Victorian waters
Document TypePhD thesis
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© 2018 Dr. Nijoy John
Cyanobacterial blooms represent a major water quality and public health issue due to their potential to produce taste and odour (T/O) compounds, and a variety of toxic metabolites. Monitoring and management of cyanobacterial blooms in various water resources is in turn dependent on the availability of accurate and sensitive diagnostic tools, and contemporary or prior knowledge of the regional distribution of toxin and T/O types. For example, in Victoria, Australia, there is limited knowledge of the distribution/ecology of geosmin producers, such that prioritising taxa for routine monitoring is difficult. Additionally, knowledge about the prevalence of many yet to be identified or emerging toxic cyanobacteria (eg., anatoxin producers) in Australia, remains poorly understood. Due to the many limitations of commonly used detection methods (i.e., microscopy and direct detection techniques), to identify toxic and T/O producing cyanobacteria, there is a need to develop sensitive and accurate diagnostic tools. PCR-based tools have shown potential to accurately identify toxic or T/O producing cyanobacteria; however, they have important limitations for routine monitoring. Although multiplex-tandem PCR (MT-PCR) tool has shown significant potential to accurately detect four major cyanotoxins (i.e., MC, NOD, STX and CYN) in Australia, it requires additional field testing and further, this tool is not presently suitable to detect anatoxin and geosmin-producing cyanobacteria. The work presented in this study used PCR-based genetic screening tools (i.e., conventional PCR, nested-PCR or MT-PCR) for accurate diagnosis of cyanotoxins and geosmin-producing cyanobacteria on environmental cyanobacterial bloom samples collected from various parts of Victoria, Australia, over a period of eight years (i.e., from 2010 to 2018). The study also employed metagenomic approaches (eg., Illumina MiSeq and PacBio) using biomarker genes [eg., 16S ribosomal RNA (16S rRNA) and phycocyanin intergenic spacer sequence (PC-IGS or cpcBA-IGS)] to elucidate the community structure of a recent (i.e., 2016) cyanobacterial bloom in the Murray River, Victoria, Australia. Based on conventional PCR-based genetic screening (using three different primer pairs) of more than 250 cyanobacteria bloom samples collected over a period of six years (i.e., 2010-2016), we detected Dolichospermum ucrainicum as the major geosmin producer in 87% of sequenced samples. Using these data, we developed a novel, small amplicon PCR primer pair capable to broadly identify all geosmin-producing cyanobacteria identified in the current study using a single standardised protocol. Additionally, genetic screening using nested-PCR on samples (n=226) collected from 2010-2017 revealed the presence and distribution of several anatoxin-producing cyanobacteria, including Cuspidothrix issatschenkoi, Aphanizomenon sp., Dolichospermum sp., at an overall sample prevalence of 30.1%. An overwhelming majority (86.8%) of anatoxin positive nested-PCR detections were from samples collected from 2016 to 2017, as compared to only 6% of samples collected prior to 2016. Using this data obtained, novel, short amplicon, PCR primers were designed for better PCR-based detection of geosmin primers. This study is the first confirming the presence of anatoxin producers in Australia. Further, field evaluation of the efficiency of MT-PCR to assess toxic cyanobacterial blooms and analysis of total community composition on samples (n=194) collected from the Murray River bloom (March 2016 – May 2016) have shown to greatly supplement microscopic examination, and highlights the potential utility of combining targeted qPCR with metagenomic amplicon sequencing methods. Based on these findings, the MT-PCR assay was expanded to detect anatoxin and major geosmin-producing cyanobacteria. The expanded MT-PCR demonstrated superior performance in comparison to conventional or nested PCRs for all but one (anatoxin) gene target in all samples (i.e., n=91 from March 2016 – February 2018) tested. Here, we report a diagnostic specificity of 100% and diagnostic sensitivity of ≥ 81% for all gene targets, and the MT-PCR platform may provide a much-needed tool for routine monitoring of toxic and geosmin-producing cyanobacterial blooms of global relevance.
Keywordscyanobacteria; cyanotoxins; geosmin; multiplex-tandem PCR; metagenomics
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