Presenter Information

Rachael Moran
David John

Location

University of South Florida St. Petersburg

Start Date

19-4-2012 12:00 AM

Description

In Gulf of Mexico, blooms of Karenia dinoflagellates cause major economic losses, wildlife mortalities, and impacting human health. Determining what Karenia species are present in a bloom, their possible succession, and implications for bloom toxicity is difficult to address due to lack of methods for rapid discrimination. Molecular methods have been developed based on Nucleic Acid Sequence Based Amplification (NASBA) of RNA that can discriminate between algal species nearly indistinguishable by microscopy. NASBA is an isothermal RNA amplification reaction amenable to field-deployment using freeze-dried reagents and a portable heat block. After characterization of novel Karenia spp. Rubisco gene sequences, multiplex NASBA primers were developed and tested, initially on in vitro transcripts from cloned genes, then on cellular RNA. Amplification products were distinguished specifically via field deployable lateral flow microarrays (LFMs). The LFM is used in a dried state, enabling extended field deployment. The LFM results for Karenia species exhibited species-specific hybridization patterns, although some cross-hybridization was apparent. Optimization of LFM hybridization stringency provided improved species-specific hybridization. The sequences of the Karenia NASBA amplicon were also used to design molecular beacons (hair-pin shaped oligonucleotides with a fluorophore and quencher molecule on either end) for use in a multiplexed detection assay capable of being read fluorometrically on a real time PCR machine. The beacons were designed such that up to 4 species could be specifically detected in a single reaction helping to provide a method of analysis for bench testing seeded samples that independent of the LFM. Beacons and LFM are being used to test seeded seawater samples and RNA purified from natural phytoplankton populations; the results of which are presented.

Comments

Presented as part of the 2012 FAS meeting as well as the 2012 USFSP Undergraduate Research Symposium held April 19, 2012.

Faculty mentor: Dr. David John and funding from: NOAA Ocean and Human Health Initiative

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Apr 19th, 12:00 AM

Development of hand-held, field deployable array biosensors to distinguish multiple Karenia species of red tide dinoflagellates.

University of South Florida St. Petersburg

In Gulf of Mexico, blooms of Karenia dinoflagellates cause major economic losses, wildlife mortalities, and impacting human health. Determining what Karenia species are present in a bloom, their possible succession, and implications for bloom toxicity is difficult to address due to lack of methods for rapid discrimination. Molecular methods have been developed based on Nucleic Acid Sequence Based Amplification (NASBA) of RNA that can discriminate between algal species nearly indistinguishable by microscopy. NASBA is an isothermal RNA amplification reaction amenable to field-deployment using freeze-dried reagents and a portable heat block. After characterization of novel Karenia spp. Rubisco gene sequences, multiplex NASBA primers were developed and tested, initially on in vitro transcripts from cloned genes, then on cellular RNA. Amplification products were distinguished specifically via field deployable lateral flow microarrays (LFMs). The LFM is used in a dried state, enabling extended field deployment. The LFM results for Karenia species exhibited species-specific hybridization patterns, although some cross-hybridization was apparent. Optimization of LFM hybridization stringency provided improved species-specific hybridization. The sequences of the Karenia NASBA amplicon were also used to design molecular beacons (hair-pin shaped oligonucleotides with a fluorophore and quencher molecule on either end) for use in a multiplexed detection assay capable of being read fluorometrically on a real time PCR machine. The beacons were designed such that up to 4 species could be specifically detected in a single reaction helping to provide a method of analysis for bench testing seeded samples that independent of the LFM. Beacons and LFM are being used to test seeded seawater samples and RNA purified from natural phytoplankton populations; the results of which are presented.