330
16.5.1.1 Denaturing Gradient Gel Electrophoresis (DGGE)
In coral bacteriology, DGGE is used to assess microbial structural differences
between coral environments such as between healthy and diseased corals, coral
water, and sediment (Fig. 16.7a). It gives a rapid fingerprint of microbial community composition, diversity, and dynamic changes over time. It is developed by
Fischer and Lerman in (1983) and is first used in microbial ecology by Muyzer et al.
(1993). It separates PCR products of dsDNA having the same length but different
sequences on a denaturant gradient polyacrylamide gel. The weaker domains of the
PCR product will begin to melt on reaching threshold denaturant concentration, and
their migration will slow down resulting in a pattern of DNA bands on the gel. Each
band on gel theoretically represents a different bacterial population present in the
community. These fingerprints can be overlapped into databases to determine bacterial fingerprint similarity or differences between similar environments in different
conditions. For example, Meron et al. (2012) used DGGE to study changes in coral
microbial communities in response to a natural pH gradient (mean pH T 7.3–8.1)
caused by volcanic CO 2 vents off Ischia, Gulf of Naples, Italy. Similarly, Ransome
et al. (2014b) used DGGE to study the difference in bacterial communities associated with healthy and diseased corals of the cold-water gorgonian coral Eunicella
verrucosa at three different sites of the southwest coast of England. They found the
stability of the bacterial community and dominance of specific genera across visibly
healthy colonies. They also found a high proportion of Endozoicomonas sequences
that has been suggested to play a role in the metabolization of dimethylsulfoniopropionate (DMSP) produced by zooxanthellae and in providing a health benefit to the
coral. They showed that diseased colonies have decreased in affiliated clones and an
increase in clones related to potentially opportunistic harmful bacteria but no
increase in a particular pathogen (Ransome et al. 2014b).
16.5.1.2 Quantitative Real-Time PCR (qRT-PCR)
In coral bacteriology, quantitative real-time PCR (qPCR) has various applications
such as pathogenic gene detection and quantification (Fig. 16.7b). It is an advanced
version of PCR that enables reliable detection and measurement of PCR product
generated during the process. It is based on the cleavage of oligonucleotide probe
that was hybridized to the target sequence, by 5′ nuclease activity of Taq polymerase during PCR process. The fluorescence signal produced by this cleavage is
used to detect amplification of the target-specific product (Heid et al. 1996). A study
by Joyner et al. (2014) used the quantitative real-time PCR for direct detection of S.
marcescens, the etiological agent of acroporid serratiosis, which is a distinct form
of white pox disease in the threatened coral A. palmate. They targeted the luxS gene
to distinguish S. marcescens from other Serratia species with a reliable quantitative
limit for the detection of ten cell equivalents (CE) per reaction.
N.P. Patel et al.
16.5.1.1 Denaturing Gradient Gel Electrophoresis (DGGE)
In coral bacteriology, DGGE is used to assess microbial structural differences
between coral environments such as between healthy and diseased corals, coral
water, and sediment (Fig. 16.7a). It gives a rapid fingerprint of microbial community composition, diversity, and dynamic changes over time. It is developed by
Fischer and Lerman in (1983) and is first used in microbial ecology by Muyzer et al.
(1993). It separates PCR products of dsDNA having the same length but different
sequences on a denaturant gradient polyacrylamide gel. The weaker domains of the
PCR product will begin to melt on reaching threshold denaturant concentration, and
their migration will slow down resulting in a pattern of DNA bands on the gel. Each
band on gel theoretically represents a different bacterial population present in the
community. These fingerprints can be overlapped into databases to determine bacterial fingerprint similarity or differences between similar environments in different
conditions. For example, Meron et al. (2012) used DGGE to study changes in coral
microbial communities in response to a natural pH gradient (mean pH T 7.3–8.1)
caused by volcanic CO 2 vents off Ischia, Gulf of Naples, Italy. Similarly, Ransome
et al. (2014b) used DGGE to study the difference in bacterial communities associated with healthy and diseased corals of the cold-water gorgonian coral Eunicella
verrucosa at three different sites of the southwest coast of England. They found the
stability of the bacterial community and dominance of specific genera across visibly
healthy colonies. They also found a high proportion of Endozoicomonas sequences
that has been suggested to play a role in the metabolization of dimethylsulfoniopropionate (DMSP) produced by zooxanthellae and in providing a health benefit to the
coral. They showed that diseased colonies have decreased in affiliated clones and an
increase in clones related to potentially opportunistic harmful bacteria but no
increase in a particular pathogen (Ransome et al. 2014b).
16.5.1.2 Quantitative Real-Time PCR (qRT-PCR)
In coral bacteriology, quantitative real-time PCR (qPCR) has various applications
such as pathogenic gene detection and quantification (Fig. 16.7b). It is an advanced
version of PCR that enables reliable detection and measurement of PCR product
generated during the process. It is based on the cleavage of oligonucleotide probe
that was hybridized to the target sequence, by 5′ nuclease activity of Taq polymerase during PCR process. The fluorescence signal produced by this cleavage is
used to detect amplification of the target-specific product (Heid et al. 1996). A study
by Joyner et al. (2014) used the quantitative real-time PCR for direct detection of S.
marcescens, the etiological agent of acroporid serratiosis, which is a distinct form
of white pox disease in the threatened coral A. palmate. They targeted the luxS gene
to distinguish S. marcescens from other Serratia species with a reliable quantitative
limit for the detection of ten cell equivalents (CE) per reaction.
N.P. Patel et al.