Early Detection of Cyanobacterial Toxins Using Genetic Methods

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Toxic cyanobacteria are typically monitored using two basic measures, cell counts and toxicity, each of which is moderately costly, highly operator dependent, and has a slow turn-around time. Rapid genetic detection of these cyanobacteria has the potential to be both faster and cheaper and provide information that complements these existing approaches. The genes that are responsible for the production of cyanotoxins can be used to design probes that will detect toxic species. This research sought to develop and test rapid genetic methods to identify toxic cyanobacteria. The key objectives were to (1) conduct a literature review and industry questionnaire examining options for rapid genetic tests, (2) characterize and understand the genes involved in cylindrospermopsin and anatoxin production, (3) adapt conventional PCR assays to real-time PCR, (4) develop rapid field methods for DNA preparation, and (5) develop probes for toxin genes and testing in the field. A comprehensive literature review and industry questionnaire were used to identify and select a suitable platform technology for rapid genetic identification of toxic cyanobacteria. The genes responsible for production of the cyanotoxins, cylindrospermopsin and anatoxin, were identified and characterized using typical molecular techniques including DNA amplification, cloning, and sequencing. The putative role of the genes was established using bioinformatics. Conventional PCR assays for the putative cylindrospermopsin toxin genes were adapted to real-time PCR by designing a gene probe for the PCR product and then optimizing the reaction. A simple field method for the extraction of cyanobacterial DNA was devised and tested after a review of relevant research. The combination of rapid extraction and real-time PCR was used to identify toxic cyanobacteria in the laboratory and field.