Methodological Approaches Used to Assess Fish Gastrointestinal Communities - Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics - page 111

Another method used to investigate microbial community composition is terminal
restriction fragment length polymorphism (T-RFLP). This method consists of the use of
fluorescence-labelled primers in the PCR amplification (usually 16S rRNA) and a subsequent
digestion of the amplicons using restriction enzymes; the fragments obtained are then
separated and detected using an automated sequence analyser (Fraher 2012). The output of
this method corresponds to different profiles (fingerprints) dependent on the composition of
the communities of the samples. T-RFLP is a fast and semi-quantitative method; however, it
does not provide phylogenetic identification and could also be affected by PCR bias.

5.2.3 Quantitative real-time PCR (qPCR)
qPCR can be used to estimate the numbers of specific bacteria in environmental samples using
specific primer sets by comparing threshold cycle (CT) T ) values with a standard curve produced
from known bacterial cell numbers plotted against CT T values. Such an approach is obviously
useful when the objective is to determine the levels of a particular bacterial species/strain. It
is useful therefore in probiotic studies and recently Avella and coworkers (2010) quantified
Lactobacillus rhamnosus recovery in the gut of clownfish ( Amphiprion ocellaris ) using this
method. A standard curve was generated based on C T values of 16S rRNA genes based on
known Lb. rhamnosus concentrations; CT T values obtained from bacterial numbers in the range
10 5 -10 9 CFU produced a linear relationship ( R 2 = 0.9914). The study demonstrated that expo-
sure of larvae and juveniles to Lb.rhamnosus via diet and/or rearing water led to high retention
of the probiotic in the GI tract, and this was later verified using the same approach in zebrafish
Danio rerio (Avella et al . 2012).
Such an approach is relatively straightforward when quantifying a known culturable strain
because accurate C T /CFU standard curves can be produced. It is however more difficult to
quantify indigenous strains of interest or total bacterial numbers in this way. He et al . (2010)
developed a qPCR method, based on the rpo B gene, to quantify the total autochthonous bac-
terial levels in the GI tract of tilapia ( Oreochromis niloticus ). The rpo B gene

was chosen because each cell contains only a single copy whereas the 16S rRNA gene poten-
tially has multiple copies per genome (e.g. seven copies in the genome of Escherichia coli )
and dominant indigenous bacteria were used to determine a standard curve between cell num-
ber and C T values. A limitation of this method is the incomplete coverage of the universal
primers and thus this approach does not truly reflect the total bacterial levels; however, this
primer set was inclusive of more than 85% bacteria in the autochthonous GI communities of
tilapia. Recently, Li etal . (2013) used qPCR to determine the relative abundance of Bacteroides
and Firmicutes in the intestinal tract of common carp ( Cyprinus carpio ) using standards con-
structed with known amounts of plasmid DNA. The method can allow for the comparison of
the relative abundance (copies) of these phyla between samples, and, when used in conjunction
with plasmid standards derived from universal primer sets, can express the proportion (%) of
the phyla with respect to total bacterial copies.

♀ × O. aureus ♂

5.2.4 Clone libraries
16S rDNA (Kim etal . 2007; Navarrete etal. 2009; Ward etal. 2009; Han etal . 2010) and cpn 60
(Mansfield et al . 2010) clone libraries have been used to evaluate the intestinal microbial com-
position in fish. PCR products derived from universal primer sets are cloned to generate bacte-
rial 16S rRNA/ cpn 60 clone libraries from each fish using commercially available cloning kits