Manning, Schonna R. , La Claire II, John .
A Multiplex PCR Method for Species-specific Detection and Quantification of the Harmful Alga, Prymnesium parvum (Haptophyta).
Harmful algal blooms have caused much concern during the past century due to an increasing frequency and severity of events. One toxin-forming alga, Prymnesium parvum, has been a nuisance since the early 1900's, causing massive fish fatalities around the globe. Due to the widespread impact of P. parvum, it is evident that sensitive methods of early detection and quantification are needed to aid the conservation of species and ecosystems affected by this ichthyotoxic alga. In this context, we have developed multiplex polymerase chain reaction (PCR) methods for rapid, species-specific detection and quantification of P. parvum. Specific cDNA sequences were examined for oligonucleotide design, and primer sets were tested using PCR to establish amplification product quality and specificity. For multiplex reactions, multiple primer sets with the required parameters were collectively tested in single reaction tubes. All simultaneously produced species-specific amplicons, as determined by gel electrophoresis and nucleotide sequencing. Reactions were performed with isolated DNA from (and, in some cases, even whole cells of) P. parvum strains from various geographic regions, including Texas, South Carolina, Maine and the United Kingdom. Related species, including other Haptophytes, and distant outgroups were utilized as negative controls. The multiplex reactions produced the same banding pattern for all P. parvum isolates examined, but this pattern was not found for any negative controls studied so far. In electrophoresis gels, we have been able to detect products from as few as ~1600 cells. Incorporating a second set of reamplification cycles increases this level of detection at least 100-fold. Continuing research is exploring the use of multiplex real-time PCR incorporating gene-specific molecular beacons, which will provide even greater sensitivity and ease of quantification. Using this assay, researchers should be able to determine the presence of this alga in environmental samples within a time frame suitable for determining appropriate responses.
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1 - University of Texas at Austin, Section of Molecular Cell & Developmental Biology, 1 University Station A6700, Austin, Texas, 78712, USA
Harmful Algal Blooms
Presentation Type: Poster
Location: Salon C, D & E - Gov Ballroom/Hilton
Date: Tuesday, August 16th, 2005
Time: 12:30 PM