Biology Reference
In-Depth Information
culture methods are time consuming. PCR (Polymerase Chain Reaction)-
based techniques, such as Denaturing Gradient Gel Electrophoresis
(DGGE) and real-time PCR, as well as non-PCR based techniques, as
Fluorescence In Situ Hybridization (FISH), have gained importance in
microbial ecology studies (Amann et al., 1990; Skovus et al., 2007, Nguyen
et al., 2007) and have been applied to different fi sh and shellfi sh species
(Griffi ths et al., 2001; Sandaa et al., 2003; Jensen et al., 2004; Goarant and
Merien, 2006; Labreuche et al., 2006; Schulze et al., 2006; Prol et al., 2009).
These methods typically rely on detection of microbial DNA to infer
information on the microbial species present in a sample and provide a
unique perspective of the components of the microbiota; however, as with
culture based methods, they also have limitations. These limitations range
from technical problems, such as obtaining representative genomic DNA
and suitable primers, to conceptual problems such as defi ning and using
meaningful taxonomic units of diversity (species) (Forney et al., 2004).
DGGE is a reliable and rapid method to study variations of dominant
bacteria and to characterize complex microbial populations (Muyzer et
al., 1993). The general principle of DGGE is the separation of fragments
of the individual rRNA genes based on differences in chemical stability or
melting temperature of the target genes. Seasonal variations of bacterial
communities associated with live preys (Rombaut et al., 2001; McIntosh
et al., 2008) and different fi sh (Griffi ths et al., 2001; Jensen et al., 2004) and
shellfi sh (Bourne et al., 2004; Payne et al., 2006; Sandaa et al., 2003) species
have been successfully characterized by using DGGE. Recently, this
fi ngerprint technique has also been used for detection and characterization
of Vibrio strains in cod larvae (Reid et al., 2009) and applied to the study of
inmunostimulants in aquaculture (Liu et al., 2008).
Real-time PCR is a specifi c and sensitive method for quantifi cation
of bacteria (Klein, 2002). This technique is based on the use of specifi c
primers and fl uorescent molecules, which permit measuring the quantity
of a specifi c amplicon at each PCR cycle. Two main chemistries have been
developed: SYBR Green, which is based on the use of a double stranded
DNA dye and TAQMAN, based on specifi c oligonucleotide probes which
have a fl uorescent dye and a quencher emitting fl uorescence only when
new copies of the target gene are created. In aquaculture, different real-
time PCR protocols have been designed, focusing on detection and
quantifi cation of pathogenic vibrios in crustaceans (Goarant and Merien,
2006), molluscs (Labreuche et al., 2006) or fi sh (Prol et al., 2009). Recently,
real-time PCR has demonstrated to be a reliable technique for specifi c
detection and quantifi cation of introduced pathogenic and probiotic
strains in fi rst feeding turbot larvae during a challenge trial (Prol et al.,
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