title: Rapid, multiplex-tandem PCR assay for automated detection and differentiation of toxigenic cyanobacterial blooms.
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.
authors.affiliation: Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors.forename: Louise
authors.initials: L
authors.lastname: Baker
authors.forename: Barbara C
authors.initials: BC
authors.lastname: Sendall
authors.forename: Robin B
authors.initials: RB
authors.lastname: Gasser
authors.forename: Toni
authors.initials: T
authors.lastname: Menjivar
authors.forename: Brett A
authors.initials: BA
authors.lastname: Neilan
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Jex
mesh_terms: D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics*; D000458:Cyanobacteria / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004784:Environmental Monitoring; D063366:Estuaries; D005618:Fresh Water / Q000382:microbiology; D005798:Genes, Bacterial; D005810:Multigene Family; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins
keywords: Cyanobacteria; High resolution melting-curve analysis; Real-time PCR; Robotic detection; Toxigenic