A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments
2010
Harmful
algal blooms(HABs) are a global problem that affects both human and
ecosystem health. One of the most serious and widespread HAB poisoning syndromes is
paralytic shellfish poisoning, commonly caused by
Alexandriumspp. dinoflagellates. Like many toxic dinoflagellates,
Alexandriumproduces resistant resting
cystsas part of its life cycle. These
cystsplay a key role in bloom initiation and decline, as well as dispersal and colonization of new areas. Information on
cystnumbers and identity is essential for understanding and predicting blooms, yet comprehensive
cystsurveys are extremely time- and labor-intensive. Here we describe the development and validation of a quantitative real-time PCR (qPCR) technique for the enumeration of
cystsof A. tamarense of the toxic North American/Group I
ribotype. The method uses a cloned fragment of the large subunit ribosomal RNA gene as a standard for
cystquantification, with an experimentally determined
conversion factorof 28,402±6152 LSU ribosomal gene copies per
cyst. Tests of DNA extraction and PCR efficiency show that mechanical breakage is required for adequate
cystlysis, and that it was necessary to dilute our DNA extracts 50-fold in order to abolish PCR inhibition from compounds co-extracted from the sediment. The resulting assay shows a linear response over 6 orders of magnitude and can reliably quantify ?10
cysts/cm3 sediment. For method validation, 129 natural sediment samples were split and analyzed in parallel, using both the qPCR and
primulin-staining techniques. Overall, there is a significant correlation (p<0.001) between the
cystabundances determined by the two methods, although the qPCR counts tend to be lower than the
primulinvalues. This underestimation is less pronounced in those samples collected from the top 1 cm of sediment, and more pronounced in those derived from the next 1–3 cm of the core. These differences may be due to the condition of the
cystsin the different layers, as the top 1 cm contains more recent
cystswhile those in the next 1–3 cm may have been in the sediments for many years. Comparison of the
cystdensities obtained by both methods shows that a majority (56.6%) of the values are within a two-fold range of each other and almost all of the samples (96.9%) are within an order of magnitude. Thus, the qPCR method described here represents a promising alternative to
primulin-staining for the identification and enumeration of
cysts. The qPCR method has a higher throughput, enabling the extraction and assay of 24 samples in the time required to process and count 8–10 samples by
primulin-staining. Both methods require prior expertise, either in taxonomy or molecular biology. Fewer person-hours per sample are required for qPCR, but
primulin-staining has lower reagent costs. The qPCR method might be more desirable for large-scale
cystmapping, where large numbers of samples are generated and a higher sample analysis rate is necessary. While the qPCR and
primulin-staining methods generate similar data, the choice of counting method may be most influenced by the practical issue of the different relative costs of labor and materials between the two methods.
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