Multiplex polymerase chain reaction (PCR) method for the rapid and sensitive species-specific detection of the harmful alga, Prymnesium parvum Carter (Haptophyta)

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Manning, Schonna Rachelle

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Harmful algal blooms have caused much concern during the past century due to an increasing frequency and severity of events. The toxin-forming alga, Prymnesium parvum Carter (Haptophyta), 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 for early detection and quantification are needed to aid the conservation of species and ecosystems affected by this ichthyotoxic alga. In this context, a multiplex polymerase chain reaction (PCR) method was developed for the rapid, species-specific detection of P. parvum. Poly-adenylated [poly-A(+)] mRNA was selected from isolated total RNA and used for cDNA library construction. Selected cDNA sequences were examined for oligonucleotide design, and primer sets were tested using PCR to establish amplicon product quality and specificity. For multiplex reactions, four primer sets with the required parameters were collectively tested in single reaction tubes. All reactions simultaneously produced four species- and gene-specific amplicons, as determined by gel electrophoresis and nucleotide sequencing. Multiplex PCR reactions were performed with isolated DNA and whole cells from P. parvum isolates from various geographic regions, including Texas, South Carolina, Maine and the United Kingdom. Isolated DNAs from related species, including other haptophytes, and distant outgroups were utilized as negative controls. The multiplex reactions resolved the same unique banding pattern for all P. parvum isolates examined and this pattern was not found for any other organisms studied so far. In electrophoresis gels, products were detectable from as few as [varies with, or, similar to]1600 cells. Incorporating a second set of re-amplification (50 cycles total) increased the level of detection at least 100-fold. Using this assay, researchers should be able to determine the presence of this toxin-forming alga in environmental samples within a time frame suitable for determining appropriate responses.



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