Detection of Invasive Species 16.6 Ecogenomic Techniques 447
Part B | 16.6
PCR Coupled Dot-Blot Hybridization
PCR coupled dot-blot hybridization involves firstly
PCR amplification and secondly detection of that amplicon using staining or radioassay (e.g.,
32 P, DIG)
of the complementary target bound to a support. The
method is useful for differentiating between closely
related species when species-specific probes are designed [16.19] and is a quick and relatively inexpensive
means of identification and discrimination. Blomster
et al. [16.19] used an ITS-based dot-blot hybridization to distinguish between species of the Chlorophyte
genus, Enteromorpha, while [16.78] employed a
32 Plabeled probe to identify Alexandrium spp.
Quantitative PCR (qPCR)
Quantitative PCR (qPCR) allows the amplification of
a target PCR amplicon to be monitored in real-time
as amplification occurs. qPCR utilizes two main detection systems: fluorescent intercalating dyes and probes.
There are a number of intercalating dyes utilized for
qPCR. The original method used ethidium bromide and
measured the change in fluorescence after each cycle
using a digital camera and a fluorometer [16.79]. Subsequent modifications incorporated intercalating dyes
such as SYBR Green I [16.80] LCGreen [16.81],
SYTO9 [16.82], and EvaGreen [16.83].
Probe-based detection systems include TaqMan
probes [16.84], molecular beacons [16.85], fluorescence resonance energy transfer (FRET) [16.86], and
minor groove binder (MGB) probes [16.87]. These
probes are more expensive than intercalating dyes but
ensure specificity of the assay through ensuring exclusive binding to the target sequence [16.82].
qPCR is typically analyzed in two ways. Relative
quantification will provide a cycle threshold (C t ) for
the specific amplification of the target sequence based
on comparison with amplification of a normalizer gene
from the same DNA template. It should be present with
the same copy number of the locus of interest. Absolute quantification is performed by determining the C t
of test samples by amplifying target DNA standards (of
known concentrations) at the same time as test samples;
the C t is calculated based on a standard curve generated
from the DNA standards.
Quantitative PCR (qPCR) (offers a relatively rapid
analysis (> 2 h)); the potential for high-throughput applications, allows linear quantification over a wide
dynamic range (> 6 orders of magnitude) and the benefit of not requiring post-PCR handling (closed-tube
format). It is now routinely used in a wide range of
clinical applications for the detection of a wide range
of bacterial, fungal, parasitic, and viral diseases of humans [16.28]. Recent advances have seen a number of
studies utilizing qPCR-based techniques for the identification of marine pests [16.72, 88].
Melting Curve Analyses. When using intercalating
dyes some qPCR thermocyclers can also perform melting analysis or high resolution melting (HRM) analysis
mutation scanning techniques. Melting analyses allows
for rapid post-PCR analysis of sequence variants or
strains/species, or a secondary confirmation of the presence of the target amplicon in the sample based on
consistency of the melt curve with the defined DNA
standards. This approach has been employed for a number of studies concerned with human and livestock
pathogens [16.89, 90]. Intercalating dyes typically bind
and allow detection of any double-stranded DNA. This
is advantageous but there is also the potential for binding to nonspecific products and primer dimers; careful
PCR optimization is required to determine the optimum
intercalating dye concentrations. The use of post-PCR
applications such as melting analysis can be utilized to
distinguish between a nonspecific product/primer dimer
and a PCR amplicon.
Potential Issues with PCR-Based Approaches
The issue of PCR inhibition, due to DNA contaminants,
potentially causing false negatives needs to be taken
into consideration for all PCR-based procedures. Incorrect pipetting techniques or aerosol contamination
of DNA and/or PCR products can contribute to false
positives in all PCR-based applications. No template
controls (NTC) should be used in every experiment to
monitor PCR contamination, and laboratories should
employ various decontamination procedures to avoid
this (e.g., Ultraviolet sterilization, decontamination of
bench tops).
16.6 Ecogenomic Techniques
Ecogenomics are methods that allow the examination
of genetic material from complex environmental samples containing many different organisms. It provides
a step-change approach to environmental surveillance
methods through its ability to identify a wider range
of taxonomic groupings [16.91]. Ecogenomic methods
Part B | 16.6
PCR Coupled Dot-Blot Hybridization
PCR coupled dot-blot hybridization involves firstly
PCR amplification and secondly detection of that amplicon using staining or radioassay (e.g.,
32 P, DIG)
of the complementary target bound to a support. The
method is useful for differentiating between closely
related species when species-specific probes are designed [16.19] and is a quick and relatively inexpensive
means of identification and discrimination. Blomster
et al. [16.19] used an ITS-based dot-blot hybridization to distinguish between species of the Chlorophyte
genus, Enteromorpha, while [16.78] employed a
32 Plabeled probe to identify Alexandrium spp.
Quantitative PCR (qPCR)
Quantitative PCR (qPCR) allows the amplification of
a target PCR amplicon to be monitored in real-time
as amplification occurs. qPCR utilizes two main detection systems: fluorescent intercalating dyes and probes.
There are a number of intercalating dyes utilized for
qPCR. The original method used ethidium bromide and
measured the change in fluorescence after each cycle
using a digital camera and a fluorometer [16.79]. Subsequent modifications incorporated intercalating dyes
such as SYBR Green I [16.80] LCGreen [16.81],
SYTO9 [16.82], and EvaGreen [16.83].
Probe-based detection systems include TaqMan
probes [16.84], molecular beacons [16.85], fluorescence resonance energy transfer (FRET) [16.86], and
minor groove binder (MGB) probes [16.87]. These
probes are more expensive than intercalating dyes but
ensure specificity of the assay through ensuring exclusive binding to the target sequence [16.82].
qPCR is typically analyzed in two ways. Relative
quantification will provide a cycle threshold (C t ) for
the specific amplification of the target sequence based
on comparison with amplification of a normalizer gene
from the same DNA template. It should be present with
the same copy number of the locus of interest. Absolute quantification is performed by determining the C t
of test samples by amplifying target DNA standards (of
known concentrations) at the same time as test samples;
the C t is calculated based on a standard curve generated
from the DNA standards.
Quantitative PCR (qPCR) (offers a relatively rapid
analysis (> 2 h)); the potential for high-throughput applications, allows linear quantification over a wide
dynamic range (> 6 orders of magnitude) and the benefit of not requiring post-PCR handling (closed-tube
format). It is now routinely used in a wide range of
clinical applications for the detection of a wide range
of bacterial, fungal, parasitic, and viral diseases of humans [16.28]. Recent advances have seen a number of
studies utilizing qPCR-based techniques for the identification of marine pests [16.72, 88].
Melting Curve Analyses. When using intercalating
dyes some qPCR thermocyclers can also perform melting analysis or high resolution melting (HRM) analysis
mutation scanning techniques. Melting analyses allows
for rapid post-PCR analysis of sequence variants or
strains/species, or a secondary confirmation of the presence of the target amplicon in the sample based on
consistency of the melt curve with the defined DNA
standards. This approach has been employed for a number of studies concerned with human and livestock
pathogens [16.89, 90]. Intercalating dyes typically bind
and allow detection of any double-stranded DNA. This
is advantageous but there is also the potential for binding to nonspecific products and primer dimers; careful
PCR optimization is required to determine the optimum
intercalating dye concentrations. The use of post-PCR
applications such as melting analysis can be utilized to
distinguish between a nonspecific product/primer dimer
and a PCR amplicon.
Potential Issues with PCR-Based Approaches
The issue of PCR inhibition, due to DNA contaminants,
potentially causing false negatives needs to be taken
into consideration for all PCR-based procedures. Incorrect pipetting techniques or aerosol contamination
of DNA and/or PCR products can contribute to false
positives in all PCR-based applications. No template
controls (NTC) should be used in every experiment to
monitor PCR contamination, and laboratories should
employ various decontamination procedures to avoid
this (e.g., Ultraviolet sterilization, decontamination of
bench tops).
16.6 Ecogenomic Techniques
Ecogenomics are methods that allow the examination
of genetic material from complex environmental samples containing many different organisms. It provides
a step-change approach to environmental surveillance
methods through its ability to identify a wider range
of taxonomic groupings [16.91]. Ecogenomic methods