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Development of a Real-Time NASBA Assay for the Detection of the Toxic Diatom Pseudo-nitzschiaEPA Grant Number: F08D20640
Title: Development of a Real-Time NASBA Assay for the Detection of the Toxic Diatom Pseudo-nitzschia
Investigators: Delaney, Jennifer
Institution: University of South Florida
EPA Project Officer: Cobbs-Green, Gladys M.
Project Period: January 1, 2008 through December 31, 2008
RFA: STAR Graduate Fellowships (2008) RFA Text | Recipients Lists
Research Category: Academic Fellowships
Biological sensor research is a newly emerging field, and one that may prove crucial for early detection of harmful algal blooms. Harmful algal blooms (HABs) have widespread negative effects on the environment, human health, and the economy. Because HABs occur along both coasts of the United States on an annual basis, it is important to have rapid and accurate methods for detecting bloom conditions. For most HAB species, current monitoring necessitates time-consuming and specialized microscopy. Molecular techniques that exploit genetic differences to discriminate toxic species are now being explored as an alternative way to monitor cell concentrations. The primary objective of this research is to design a molecular assay for the detection and enumeration of the toxic marine diatom Pseudo-nitzschia. Many species within this genus are known producers of the neurotoxin domoic acid, the causative agent of Amnesic Shellfish Poisoning and a compound responsible for wildlife mortality. Also crucial to the success of molecular assays is their ability to be incorporated into ocean monitoring platforms, and another eventual objective of this research is to integrate the Pseudo-nitzschia assay into the Autonomous Microbial Genosensor (AMG), an autonomous buoy system developed at the University of South Florida.
The molecular detection method used is nucleic acid sequence-based amplification (NASBA), which is an isothermal RNA amplification method for real-time quantification based on the fluorescence signal of a specific molecular beacon measured over time. Because of its isothermal nature, this molecular technique is ideal for integration into field-based monitoring platforms. P. multiseries, an extensively studied species that is known to be very toxic, was used as the first target Pseudo-nitzschia species. A NASBA assay was designed based on the rbcS sequence of P. multiseries, which encodes for the small subunit of the carbon fixation enzyme, RuBisCO. Two subsequent assays, one specific to P. delicatissima and one specific to a currently unidentified isolate from Louisiana waters, were also designed.
Variation in the rbcS gene has enabled the discrimination of Pseudo-nitzschia from other phytoplankton species, as well as some degree of discrimination between Pseudo-nitzschia species. The P. multiseries assay demonstrates excellent linearity (r2 > 0.99) over four orders of magnitude and sensitivity to one cell in culture. Four strains of P. multiseries were detected by this assay, as well as six out of ten strains representing other species of Pseudo-nitzschia. The additional assays for P. delicatissima and the Louisiana isolate appear to be species-specific and also demonstrate the ability to detect one cell. Testing with 33 species of non-target phytoplankton cultures (including the closely-related diatom genus Nitzschia) has resulted in no cross-reactivity of the three assays. Analysis of environmental samples, including local samples collected in Tampa Bay, FL and samples from two Gulf of Mexico cruises, is currently underway. The ability to rapidly and autonomously monitor levels of P. multiseries, and potentially other species of Pseudo-nitzschia,will result in preventative strategies that benefit both human and environmental health.