Evaluation of Nucleic Acid Probes for....
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GRANT NUMBER:  NA57FD0009           NMFS NUMBER:  93-SWR-007

REPORT TITLE:  Evaluation of Nucleic Acid Probes for Identifying Domoic Acid-Producing Pseudo-nitzschia Species

AUTHOR:  Scholin, Christopher A.

PUBLISH DATE:  July 15, 1996

AVAILABLE FROM:  National Marine Fisheries Service, Southwest Region, 501 West Ocean Blvd., Suite 4200, Long Beach, CA 90802-4213.   PHONE: (562) 980-4033


Some but not all marine pennate diatoms of the genus Pseudo-nitzschia are associated with the production of domoic acid, the causative agent of amnesic shellfish poisoning (ASP).  Distinguishing between potentially toxic and nontoxic representatives of this genus is time-consuming and often difficult. In an effort to speed and ease species identifications a suite of ribosomal RNA-targeted DNA probes were devised for identifying P. americana, P. fraudulenta, P. heimii, P. pungens, P. australis, P. delicatissima, P. multiseries and P. pseudodelicatissima.  Of those species the latter four as they occur in North America are considered potentially toxic, with the greatest public health threat likely to arise from blooms of P. australis, P. multiseries and P. pseudodelicatissima.  Flourescently labeled probes for all species were applied using a filter manifold system with sequential addition of reagents; the resulting filters were transferred to a conventional microscope slide and viewed using epiflourescence microscopy. Labeled cells were enumerated and by knowing the volume of water filtered it was possible to estimate the abundance of particular Pseudo-nitzschia species in cultured and field samples.  A colorimetric "dipstick-type" assay was also devised for detection of P. australis, P. multiseries, P. pseudodelicatissima and P. pungens in a single sample, simultaneously, using a semi-automated processor. This portion of the work was done in collaboration with MicroProbe Corp. In this case, cells from cultured or field samples were collected onto a filter then lysed (homogenized).  The cell lysate was transferred to a custom instrument for automated processing. The end result was a colorimetric reaction, the intensity of which was proportional to the abundance of targeted species in the original sample.  Both methods of probe application (whole cell fluorescence and dipstick colorimetry) are promising and rapid means by which one might detect and quantify Pseudo-nitzschia species as they occur in natural samples.  Neither method is technically demanding nor expensive.  More work is required to test the utility of the methods for routine plankton monitoring and their applicability for detecting a wider range of aquatic-borne species that are of public health or economic concern.

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