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Mol Cell Probes. 2013 Oct-Dec;27(5-6):208-14. doi: 10.1016/j.mcp.2013.07.001. Epub 2013 Jul 10.

Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms.

Author information

1
Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia.

Abstract

Cyanobacterial blooms are a major water quality issue and potential public health risk in freshwater, marine and estuarine ecosystems globally, because of their potential to produce cyanotoxins. To date, a significant challenge in the effective management of cyanobacterial has been an inability of classical microscopy-based approaches to consistently and reliably detect and differentiate toxic from non-toxic blooms. The potential of cyanobacteria to produce toxins has been linked to the presence of specific biosynthetic gene clusters. Here, we describe the application of a robotic PCR-based assay for the semi-automated and simultaneous detection of toxin biosynthesis genes of each of the toxin classes characterized to date for cyanobacteria [i.e., microcystins (MCYs), nodularins (NODs), cylindrospermopsins (CYNs) and paralytic shellfish toxins (PSTs)/saxitoxins (SXTs)]. We demonstrated high sensitivity and specificity for each assay using well-characterized, cultured isolates, and establish its utility as a quantitative PCR using DNA, clone and cell-based dilution series. In addition, we used 206 field-collected samples and 100 known negative controls to compare the performance of each assay with conventional PCR and direct toxin detection. We report a diagnostic specificity of 100% and a sensitivity of ≥97.7% for each assay.

KEYWORDS:

Cyanobacteria; High resolution melting-curve analysis; Real-time PCR; Robotic detection; Toxigenic

PMID:
23850895
DOI:
10.1016/j.mcp.2013.07.001
[Indexed for MEDLINE]

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