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Keyword: DNA detection; Dreissena; polymerase chain reaction;
Laser transmission spectroscopy; ballast; invasive species
2013.02.07 정다금
Abstract
http://en.wikipedia.org/wiki/Quag
ga_mussel
Status:
- Early detection of invasive species is important
Solution:
- Laser transmission spectrosocpy(LTS) is a powerful solution
offering real-time DNA based species detection in the field
How:
- measure the size, shape, number of nanoparticles in a solution
- From hybridization of the pcr product to nanoparticles
(with species-specific oligonucleotide probes or probes alone)
What:
- invasive freshwater quagga mussel(Dreissena bugensis)-indigenous to Ukraine
Purpose:
- To evaluate the capability of the LTS platform for invasive species detection.
Test:
1) DNA concentrations of a single target species
2) The presence of a target species within a mixed sample
of other closely related species
3) Species-specific functionalized nanoparticles vs.
species-specific oligonucleotide probes alone
4) Amplified DNA fragments vs. unamplified genomic DNA
-> LTS is a highly sensitive technique for rapid target species detecetion
- Detection limits: picomolar range.
- Capable of successful identification in multispecies samples with target and nontarget species DNA
Result:
LTS DNA detection platform will be useful for field application of target species.
LTS have versatility for broader application: effective with species-specific
oligonucleotide tags alone or with polystyren nanobeads, with both amplified and
unamplified DNA.
Introduction
Invasive species:
Problem
- Negative effects on ecosystem(fresh,marine), biodiverisy, commerce
- Economic damage: $120 billion annually for the USA
(ex: Laurentian Great Lakes: 180 species are introduced via ships’ ballast
Great Lake: 150million damage annually in power plants.):
How to solve: Need for rapid and inexpensive field-based detection technologies
to identify harmful species in water samples from ballast water etc.
before establishment or spread
Previous studies
Research on detecting rare species:
-Focus on environmental samples
-Detection of aquatic organisms
-Species identification: Relies on forensic DNA evidence
Novel platforms for early detection
- Fluorescence
- Nanotube chips
Strength:
higher specificity, fast relative to traditional method of hand sorting and
morphologically based microscopy
Weakness:
- high costs, low throughput, lengthy detection times
(e.g chip fabrication time and sample screening: 2-6h))
-Dependence on technical expertise for platform
Need:
-more efficient method(field based detection, rapid analysis and quick decision)
--> Laser transmission spectroscopy(LTS)
- provide rapid, cost-effective, user friendly detection of invasive species
Figure. Schematic diagram of DNA detection using LTS.
Concept
-Measures wavelength-dependent light transmittance through a sample
containing nanoparticles in suspension
- The transmission of light through the sample cell containing paritcles plus
suspension fluid is recorded along with that of a similar cell containing only
the suspension fluid.
-The data are analysed and inverted by a computer algorithm that outputs
particle size distribution(X axis) and abundance(Y axis).
LTS
-Quantitative detection platform
-Measuring the size and shape
and number of nanoparticles in a
solution
-Binding-> increase in particle
size
A resolution of 3nm for
mixtures/ 1nm -> 0.3-1 DNA
base pair
=>Differentially sized molecules
produce different peak profiles,
depending on their size and
concentration in solution.
-> species specific tags
-> LTS peak is diagnostic for the
detection of an intended target
-> differ by 7bp in a 32 bp
species-specific region.
ssDNA
nanobead
Tag
Figure. Schematic diagram of nanobead preparation (Steps 1 and 2) and
binding of DNA to the functionalized beads (Steps 3–5).
DNA prep: amplified DNA- denature(2min)-incubated with tagged
nanobeads
1.1 Rationale
Q about the efficacy of LTS for application in field settings.
1) What are the concentration limits for detection using the LTS platform?
2) How is detection affected by the presence of non-target DNA(DNA from
other species?)
3) Can LTS-based detection occur without the use of nanobeads
(i.e. with only the DNA and species-specific tags present)?
4) Can LTS-based detection occur in the presence of unamplified genomic
DNA?
1. Condition1: PCR amplified DNA and with genomic DNA
2. Condition2: species-specific oligonucleotide-tagged nanobeads
and only species-specific oligonucleotide tags
2. Materials and Methods
2.1 Sample preparation: DNA extraction and amplification
Target species: quagga mussel- Q
Non-target species:
1.Zebra mussel(Dreissena polymorpha) - ZM
2. Golden mussel(Limnoperna fortunei),-GM
3. Chinese mitten crab(Eriocheir sinensis),-CM
4. water flea(Daphnia magna))-DM
-
Genomic DNA extraction
PCR(COI gene 600bp)
Quantification (gDNA and PCR product)
Purification
2.2 Testing the limits of detection for the laser transmission spectroscopy platform
under optimal conditions
•Serial dilution (n = 5), 1 to 2.5 * 10-5 ng/ul
•Blind test(no bias in intepretation of results)
•Negative control: Solutions with no DNA
• initial-taged bead control, and four dilutions) -> 5 measurements.
2.2 Testing the limits of detection
for th eLTS platform
under optimal conditions
- Tagged nanoparticles hybridized
to target species DNA had larger
diameters than tagged
polystyrene beads alon at all
concentration.
- Shifts in diameter due to ‘target
DNA detection’
The relationship between
concentration and the position of
the LTS peak
2.3 Testing laser transmission spectroscopy detection in mixed samples
with tagged beads using PCR product or genomic DNA
•
•
•
•
Target species only
Target and multiple non-target species
Non-target species only.
Used simulated ballast samples : typically 0.2-2.0 ng/ul, Blind test two times.
2.3 Testing laser transmission spectroscopy detection in mixed samples
with tagged beads using PCR product or genomic DNA
Peak shift of 16-18nm
Peak
shift of
34nm
Minimum peak
shift of 16nm:
less than
1/100th of a
percent(z-test)
2.4 Testing laser transmission spectroscopy detection in mixed samples
with no beads using PCR product or genomic DNA
• Could reduce processing time?
• by eliminating beads from the assay.
•Potentially offer significant risk reduction.
• bead: polystyrene- susceptible to heat and cold damage., limited lifetime.
2.4 Testing laser
transmission
spectroscopy
detection in mixed
samples with
no beads using PCR
product or genomic
DNA
PCR product
gDNA
4. Discussion
Real-time information about the presence and distribution of target
species is necessary for shipping industry operators or government
agencies to respond to the growing threat of biological invasions in
aquatic environments.
Detection tools: rapid and accurate, easy to use
in both application and interpretation of results
LTS
- Using regression model, Concentration range for potential detection to the
picomolar(10-12)range (y=0)
-Ultimately to screen DNA samples collected directly from nature w/o PCR
-Collection to finalanalysis (3hr, including PCR) -> under 1hr (w/o PCR)
- To train ship crew members while the ship is underway
- cost effective and easily applied platform (sample processing and analysses
costs are under 5$ per sample..
To be resolved
- To apply for many environmental DNA(eDNA) based samples, further tests
will be necessary to determine whether our results are generalizable to other
species.
- Samples could be screened for broader taxonomic groups.
- Only a few amplification cycles may be needed for detection (if PCR is required)
- To test different sized polystyrene beads with different oligonucleotide tags
- Quality assurance for detections (graduate to real-time testing of samples on
site in the field)
Conclusion
Current study: The potential usefulness and versatility of LTS technology for
species-specific DNA detection in aqueous samples, and for particular
application to ship ballast water samples.
By detecting invaders early by use of the LTS platform, management and
governmental groups can rapidly respond at stages where establishment can
potentially be prevented or more efficiently managed.