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
ABSTRACT

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.