Molecular Detection of Karenia spp. in the
Mid-AtlanticKathryn J. Coyne1, Edward Whereat1, Muns Farestad1, Jennifer Torora1, Lauren Salvitti1 and Carmelo Tomas2
1University of Delaware College of Earth, Ocean, and Environment
2University of North Carolina, Center for Marine Science
Interstate Seafood Seminar, Sept. 16, 2010
OverviewOverview
• Molecular approach – Description– Rationale– Development– Validation
• Karenia spp. in Mid-Atlantic coastal waters– Timeline– Molecular (presence/absence) results– Validation and results of quantitative approach for
enumeration– Morphological variability– Distributions of Karenia spp. in DE and NJ
What are Molecular Methods?What are Molecular Methods?
C C C CCT AA GG G GT T T T T T TT AA
C C CCT AA GG GT T T T T T TT AAA A
C C C CCT AA GG GT T T T TT AAA AG
Molecular MethodsMolecular Methods• Polymerase chain reaction (PCR)
– Specific amplification of DNA fragment from a specific gene
DNA primers bracket the area to be amplified
Polymerase enzyme copies DNA fragment bracketed by primers to produce a product of a single size.
Why do we use Molecular Methods for HAB Research?
To determine presence/absence or abundance of HAB speciesAdvantages over microscopy:
– Increased sensitivity– Increased accuracy in detection– Applicable to mixed assemblages– Higher throughput
Species are identified and discriminated based on variations in DNA sequence.
rRNA genes - 10s to 100s of copies
Highly conserved regions
-Group or genus-specific primers
Variable regions
-Species or strain-specific primers
18S small subunit ITS1 5.8S ITS2 28S large subunit
Quantitative Real-Time PCR
Wide dynamic range: Linear over 8 orders of magnitude
Sensitive: Detection of less than 4 copies of target DNA is possible
Ct
Yes
How do we validate PCR methods?
Design primers for target species or
group
Use primers to amplify DNA from several samples
Sequence DNA to make sure primers
are specific
STEP 1: Primer design
YesNo
STEP 2: QPCR assay development
Optimize primers for QPCR
Evaluate sensitivity and range of detection
Determine accuracy
Cell counts
QPCR results
Research and
Monitoring
No
2008June - Nov., 2008: Water samples (n=33) from 4 sites in DE processed for molecular analysis. 2009Mar. - Nov., 2009: Water samples (n=30) from 4 sites in DE processed for molecular analysis. Aug. 26-27: Water samples (n=7) from NJ coastal bays processed for molecular analysis.2010March - present, 2010: Water samples from 4 sites in DE processed for molecular analysis.
2007August 30, 2007: Initial observation of K. papilionaceae in water samples.Aug. - Sept., 2007: Microscopic identification of K. pap in off-shore waters and ocean beaches.Sept. 6 - 7, 2007: Smaller “K. brevis -like” cells observed in water samples.
Karenia Karenia spp. in Mid-Atlanticspp. in Mid-Atlantic
Primer design and evaluation (Kpap)Primer design and evaluation (Kpap)
Primers for K. pap were initially provided by Miguel de Salas (UTasmania)
•Targets the 28S rRNA gene.
•DNA was amplified by PCR from environmental samples collected Aug.-Sept., 2007.
•Sequenced PCR products from 3 env. samples collected on 2 different days.
•Comparison of DE strain of Kpap with Kpap sequences in GenBank from Hong Kong, NZ and Australia
Results: Primers are specific to Kpap, but sequence is slightly different
Later confirmed by sequence analysis of pure culture of NZ and DE strains provided by Dr. Carm Tomas (UNC)
M 1 2 3 N
When did When did K. papilionaceae K. papilionaceae first appear first appear in DE coastal waters?in DE coastal waters?
• DNA from inlet and Fenwick beach water samples collected in June and July, 2007 were tested.
• K. pap was not present on June 25, 2007.• Samples collected on July 11, 2007 through the
end of August were positive.
Results suggest that K. pap was present early in the summer
K. pap K. pap PCR Screening (presence/absence) PCR Screening (presence/absence) for DE coastal waters 2008-2009for DE coastal waters 2008-2009
• 2008: 33 samples from 4 sites. – 13 samples collected before Aug. 14th were negative for K.
papilionacea– All 20 samples collected between Aug. 14th and Nov. 13th were
positive for K. pap.
• 2009: 33 samples from 4 sites. – 16 samples collected before Aug. 17th were negative for K.
papilionacea– 1 sample collected on Aug. 3rd was positive (IR39)– All 14 samples collected between Aug. 17th and Nov. 4th were
positive for K. papilionacea
• 2010: Samples collected Mar. through present– Samples collected before Aug. were negative for K. pap
DE isolate genomic DNA
DE isolate positive control
NZ isolate positive control
DE isolate pos cont and genomic DNA
NZ isolate positive control
QPCR assay for QPCR assay for K. papilionaceaeK. papilionaceae
New primers and QPCR probes were designed based on sequence data
- Specific to NZ and DE strains
1
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Date and Location
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QPCR
cell count
QPCR Results for QPCR Results for K. papilionaceaeK. papilionaceae 2008 Samples 2008 Samples
Cell Count Detection limit = 167 cells/L
• QPCR results are comparable to cell counts when above limit of detection
• QPCR is more sensitive than cell counts
Calibrator sample
M 1 2 3 4 5 6 7 P N
Distribution of Distribution of K. pap K. pap north of Delawarenorth of Delaware
Samples collected along NJ on Aug. 26-27, 2009
K. pap was present from Cape May to Ocean City
Molecular detection of Molecular detection of K. brevisK. brevis
• Culture obtained from CCMP to use as positive control• 2 Different primer sets designed targeting 28S rRNA gene• Primers for K. brevis RUBISCO (Gray et al. 2003)• PCR optimized using DNA from K. brevis culture
Results: Environmental samples do not amplify with the 3 different K. brevis primer sets
- Weak, non-specific amplification
M 1 2 3 N 1 2 3 N Kpap Kbrev
Morphological Variability Morphological Variability • Unialgal cultures of the DE strain of K. papilionaceae
were established by Dr. Carm Tomas.• Small “K. brevis –like” cells were observed in these
cultures.• Small and large cells were picked into separate tubes
and sent for DNA analysis.
Results: Both large and small cells amplified with K. pap primers but not with K. brevis primers.
It’s possible that the “K. brevis –like” cells in environmental water samples were actually K. papilionaceae.
Another possibility is that the “K. brevis –like” cells in environmental water samples were an unknown Karenia strain or species.
More work needs to be done
Summary and ImplicationsSummary and Implications
• K. papilionacea has been detected in DE coastal waters each year since 2007– Endemic population or imported each year?
• K. brevis-like cells have been observed but have not been detected using molecular methods– Morphological variability of K. papilionaceae
or new strain/species of Karenia?