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Molecular-Based Research
and Monitoring
 The
molecular research arm of the SCAEL develops
species-specific real-time PCR assays for the detection of harmful
algae from
environmental samples. A Smart
Cycler®
(Cepheid), an integrated real-time PCR amplification and detection
system, has
been used in the development of real-time PCR assays. This technology
combines
amplification and detection into one system. Rapid, precise heating and
cooling
of reaction mixtures allows for decreased assay times and increased
productivity. A dual-labeled, internal fluorescent oligonucleotide
probe
enhances assay specificity over traditional PCR. Fluorescence detection
is used
to measure amplification products as they are generated in
real-time.
Currently,
eight different assays
targeting Karlodinium micrum
(Tengs et al. 2001), Pfiesteria
piscicida, P. shumwayae
(Bowers et al. 2000), Chattonella
verruculosa, and C.
cf verruculosa, (Bowers
unpub.), Heterosigma
akashiwo, Fibrocapsa
japonica,
and Kryptoperidinium
foliaceum (Kempton
unpub.) are used for monitoring and research efforts in the
SCAEL. These
assays have been integrated into existing monitoring efforts by
confirming all
presumptive positives for the species listed. Samples are identified
based on
light microscopy immediately after collection, and then confirmed using
the
species-specific assays. In addition to routine monitoring, the
real-time PCR
assays
are also used to confirm the identity of target organisms from fish
kill events, and from ongoing in situ nutrient and oyster
bioassays. Assays for Chattonella
subsalsa, Aureococcus anophagefferens, and Microcystis
aureginosa are
currently being integrated into our existing monitoring network. In
addition to
real-time PCR, fluorescent in situ hybridization (FISH) assays
for P.
piscicida (Rublee et al. 1999) and K. foliaceum (work in
progress)
are being used to identify and quantify target organisms from sediment
samples
The RT-PCR
technology has also been modified to include
direct RT-PCR. The existing assays can now be taken directly from the
field and
tested without having to use a timely extraction protocol. Hand-picked
cells
and concentrated cells can be placed directly into a reaction tube, and
amplified accordingly if the target sequence is present. Blooms and
fish kill
samples can now be confirmed for the presence of harmful algal species
(if the
assays are available) in as little as 75 minutes. Direct PCR thus far
has been
used to confirm a PLO bloom (1.7 X 104 cell ml-1)
in a
brackish detention pond on Kiawah Island, SC as Pfiesteria piscicida and confirm the identity of
Heterosigma
akashiwo from a fish kill off of Bulls Bay, SC .
In
addition to assay development, future efforts will focus on determining
cyst
populations of several harmful species. Sediment samples were collected
from Bulls Bay, SC
over a large spatial
scale to determine the presence of H. akashiwo cysts . The H.
akashiwo bloom (9.3
X 104
cell ml-1) that was noted on 29 April 2003 is estimated to have covered at
least 80 sq. mi.
Preliminary data suggests that the bloom may have originated inshore.
The real-time PCR assay for H.
akashiwo will be used to evaluate the
spatial extent
of H. akashiwo cysts residing in the inshore sediments (Bulls
Bay and adjacent
creeks). The Pfiesteria
piscicida real-time PCR assay has been used to track the
distribution of P.
piscicida in the tidal creeks and rivers adjacent the Kiawah
Island, SC detention ponds . P.
piscicida has
been
detected in the sediments of 35 out of 59 ponds and 11
out of 42 creek and river samples on Kiawah Island.
If these ponds are acting as “natural incubators” for the promotion of
HAB
species, it is important to also understand the extent these harmful
algal
species are getting transported into adjacent waters.
All content © 2003 S.C.A.E.L. any
use of contents
requires permission from the Webmaster.
Algal Ecology Labs--331 Fort Johnson
Road--Charleston,
SC 29414.
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