Detection of Invasive Species 16.4 Molecular Approach to the Identification of Marine Invasive Species 443
Part B | 16.4
Effective routine detection and surveillance of marine pests from environmental samples requires effective sample collection and genomic DNA (gDNA)
isolation methodology. Bott et al. [16.11] highlighted
the need to use samples that will be biologically informative for surveillance purposes. Organisms can be
collected from water by filtering known amounts and
extracting DNA from the filtrate. Larger sample sizes
increase the likelihood of containing invasive species
but pose issues surrounding DNA extraction.
Inhibition of PCR due to contaminants in environmental samples can be a limiting factor to accurate
detection of invasive species. PCR inhibitors include organic and phenolic compounds, fats, glycogen, Ca
2C ,
humic acids, heavy metals, constituents of bacterial
cells, over-abundance of nontarget DNA, and other
contaminants [16.17]. There is a need, therefore, to rigorously assess DNA extractions for the presence of PCR
inhibitors to reduce the occurrence of false negatives
during analysis. Screening DNA extracts with control PCR assays of a known, added indicator organism
is a useful approach in samples containing numerous DNA types. Correct preparation of environmental
samples for DNA extraction can also prove useful in
preventing PCR inhibition. For example, anecdotal evidence of freeze-drying marine sediment samples prior
to DNA extraction greatly reduces the effects of PCR
inhibition [16.11], which may be due to increased DNA
extraction efficiency in freeze-dried samples [16.18].
A simple option to limit the effects of PCR inhibition
is through the dilution of DNA; provided that a reliable
detection limit to detect an organism is known, samples
can be diluted to reduce inhibitors to below critical concentrations.
16.4 Molecular Approach to the Identification of Marine Invasive Species
As the use of molecular biological techniques has become more widespread, so too has their use in the
identification of marine invasive species. A range of
DNA-based techniques have been used (Table 16.1)
to discriminate between species because of the ability
of these methods to detect subtle genetic differences.
DNA is relatively stable and can be readily isolated
from fresh, frozen, ethanol-preserved, or commercially
available nucleic acid storage buffer (e.g., RNAlater©,
Ambion) preserved specimens for subsequent analysis
using cloning, hybridization and enzymatic amplification procedures.
PCR [16.58, 59] is a reliable method of enzymatic
nucleic acid amplification which has significantly advanced a wide range of scientific areas. The majority of
molecular assays for the detection and identification of
marine invasive species and their allies involve PCRbased technologies. The key to a reliable and robust
molecular method for the identification of a marine pest
is the definition of one or more suitable DNA target regions (genetic marker or locus).
Marine invasive species encompass a range of taxonomic groupings and no single marker should be
considered universally appropriate to all taxa. Nuclear
ribosomal genes and spacers, mitochondrial genes, and
chloroplast genes (for plants), have been utilized to
identify marine invasive species (Table 16.1). A thorough knowledge of the sequence variation within
the marker of choice (both intra and inter-specific
variation) must be obtained from previously published data (e.g., GenBank- http://www.ncbi.nlm.nih.
gov/Genbank/). DNA sequencing of the marker of
choice of both target and related organisms must be carried out in order to design an assay which is specific to
the target taxon.
Genes evolve at different rates and a suitable DNA
region should vary in sequence sufficiently to allow the
identification of an individual to the taxonomic level
required. For specific identification, the DNA marker
should exhibit little or no genetic variation within
a species but differ sufficiently between species so as
to allow unequivocal delineation. For the identification
of population variants (strains or genotypes), the marker
should vary considerably in sequence within a species
but still offer enough specificity so as to not cross-react
(false-positives) with heterologous species.
In nuclear genes and spacers, there is typically little variation amongst individuals within a population
and between other populations [16.43, 45]. Nuclear ribosomal DNA (rDNA) of eukaryotes is repetitive and
consists of hundreds of tandem repeats. These tandem repeats are often distributed on different chromosomes [16.60, 61] and like other repetitive DNA
elements they show sequence homogeneity as a result
of concerted evolution [16.61, 62]. The rDNA array
consists of the intergenic nontranscribed spacer (IGS
or NTS), the external transcribed spacer (ETS), and the
transcription unit comprising three rRNA genes (short
subunit (SSU), 5:8 S and long subunit (LSU)), which
are separated by the first and second internal transcribed
Part B | 16.4
Effective routine detection and surveillance of marine pests from environmental samples requires effective sample collection and genomic DNA (gDNA)
isolation methodology. Bott et al. [16.11] highlighted
the need to use samples that will be biologically informative for surveillance purposes. Organisms can be
collected from water by filtering known amounts and
extracting DNA from the filtrate. Larger sample sizes
increase the likelihood of containing invasive species
but pose issues surrounding DNA extraction.
Inhibition of PCR due to contaminants in environmental samples can be a limiting factor to accurate
detection of invasive species. PCR inhibitors include organic and phenolic compounds, fats, glycogen, Ca
2C ,
humic acids, heavy metals, constituents of bacterial
cells, over-abundance of nontarget DNA, and other
contaminants [16.17]. There is a need, therefore, to rigorously assess DNA extractions for the presence of PCR
inhibitors to reduce the occurrence of false negatives
during analysis. Screening DNA extracts with control PCR assays of a known, added indicator organism
is a useful approach in samples containing numerous DNA types. Correct preparation of environmental
samples for DNA extraction can also prove useful in
preventing PCR inhibition. For example, anecdotal evidence of freeze-drying marine sediment samples prior
to DNA extraction greatly reduces the effects of PCR
inhibition [16.11], which may be due to increased DNA
extraction efficiency in freeze-dried samples [16.18].
A simple option to limit the effects of PCR inhibition
is through the dilution of DNA; provided that a reliable
detection limit to detect an organism is known, samples
can be diluted to reduce inhibitors to below critical concentrations.
16.4 Molecular Approach to the Identification of Marine Invasive Species
As the use of molecular biological techniques has become more widespread, so too has their use in the
identification of marine invasive species. A range of
DNA-based techniques have been used (Table 16.1)
to discriminate between species because of the ability
of these methods to detect subtle genetic differences.
DNA is relatively stable and can be readily isolated
from fresh, frozen, ethanol-preserved, or commercially
available nucleic acid storage buffer (e.g., RNAlater©,
Ambion) preserved specimens for subsequent analysis
using cloning, hybridization and enzymatic amplification procedures.
PCR [16.58, 59] is a reliable method of enzymatic
nucleic acid amplification which has significantly advanced a wide range of scientific areas. The majority of
molecular assays for the detection and identification of
marine invasive species and their allies involve PCRbased technologies. The key to a reliable and robust
molecular method for the identification of a marine pest
is the definition of one or more suitable DNA target regions (genetic marker or locus).
Marine invasive species encompass a range of taxonomic groupings and no single marker should be
considered universally appropriate to all taxa. Nuclear
ribosomal genes and spacers, mitochondrial genes, and
chloroplast genes (for plants), have been utilized to
identify marine invasive species (Table 16.1). A thorough knowledge of the sequence variation within
the marker of choice (both intra and inter-specific
variation) must be obtained from previously published data (e.g., GenBank- http://www.ncbi.nlm.nih.
gov/Genbank/). DNA sequencing of the marker of
choice of both target and related organisms must be carried out in order to design an assay which is specific to
the target taxon.
Genes evolve at different rates and a suitable DNA
region should vary in sequence sufficiently to allow the
identification of an individual to the taxonomic level
required. For specific identification, the DNA marker
should exhibit little or no genetic variation within
a species but differ sufficiently between species so as
to allow unequivocal delineation. For the identification
of population variants (strains or genotypes), the marker
should vary considerably in sequence within a species
but still offer enough specificity so as to not cross-react
(false-positives) with heterologous species.
In nuclear genes and spacers, there is typically little variation amongst individuals within a population
and between other populations [16.43, 45]. Nuclear ribosomal DNA (rDNA) of eukaryotes is repetitive and
consists of hundreds of tandem repeats. These tandem repeats are often distributed on different chromosomes [16.60, 61] and like other repetitive DNA
elements they show sequence homogeneity as a result
of concerted evolution [16.61, 62]. The rDNA array
consists of the intergenic nontranscribed spacer (IGS
or NTS), the external transcribed spacer (ETS), and the
transcription unit comprising three rRNA genes (short
subunit (SSU), 5:8 S and long subunit (LSU)), which
are separated by the first and second internal transcribed