Graduation Year

2004

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Marine Science

Major Professor

Paul, John H.

Keywords

rbcL, real-time PCR, red tide, harmful algal bloom, monitoring, Gulf of Mexico, Gymnodinium breve

Abstract

Karenia brevis (Davis cf. Hansen & Moestrup = Gymnodinium breve) is the non-peridinin containing dinoflagellate responsible for many harmful algal blooms (red tides) in the Gulf of Mexico. These recurrent blooms can have significant negative ecological, economic, and human health impacts including fish kills, tainting of shellfish, poisoning of marine mammals, loss of tourism revenue due to beach closures, and respiratory distress and food poisoning in humans. A method for detection of Karenia brevis was developed based upon amplification of the mRNA for the plastid-encoded gene of the carbon fixing enzyme ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) large subunit (rbcL). Using sequence information from a primer set targeting a 554-bp region of the Karenia rbcL gene, a small (91 bp amplicon) primer and probe set was created for TaqMan(registered trademark) real time RT-PCR of K. brevis rbcL.

The primer/probe set is sensitive to as little as 0.1 fg of target transcript and as little as 1 pg of total cellular K. brevis RNA extract, corresponding to less than 1 cell reaction-1. The primer/probe set did not amplify rbcL transcript from any of the non-target algae tested. Bloom samples analyzed by this method have shown the assay to be a reliable method, with effective enumeration and a linear relationship showing good correlation to the cell counts by microscopy (r2= 0.8344). The assay has been shown to be robust and perform well even in non-ideal conditions, with pre-extraction RNA from unialgal culture stable at room temperature for up to 3 days and up to a month at -80 degrees C in Stratagene's lysis buffer. The transcription of the rbcL gene demonstrated minor variation throughout the diel period, however the variation was not linked to the diel cycle or to carbon fixation, which showed a distinct diel signal.

Due to the relatively constant expression of the rbcL gene, the real-time RT-PCR assay developed should be able to reliably enumerate K. brevis populations in the natural environment, as long as the sample is placed in Stratagene's lysis buffer and processed within one or two days or frozen at -80 degrees C and processed within a month.

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