170 7 Pollution of Aquatic Systems: Pollution Through Eutrophication, Fecal Materials, and Oil Spills
genome. The large genomic fragments are
placed in a unique gel apparatus where electric
current is passed through a gel in different
directions at low voltage for 10–12 h to achieve
the best level of band separation possible. PFGE
is similar to ribotyping, but instead of analyzing
rRNA, it uses the whole DNA genome.
In another system of PFGE, the bacterial
DNA is embedded in an agarose plug and
digested while in the plug; the plugs are placed
in hollow gel combs and become part of the gel
as the gel is cast around the combs. The gels are
stained and photographed after electrophoresis.
It is better for discriminating host sources
compared to the other fecal coliforms.
(e) Amplified fragment length polymorphism
(AFLP-PCR)
Amplified Fragment Length Polymorphism
(AFLP) is a polymerase chain reaction (PCR)
based genetic fingerprinting technique. AFLP
uses restriction enzymes to cut genomic DNA,
followed by ligation of complementary double
stranded adaptors to the ends of the restriction
fragments. A subset of the restriction fragments
are then amplified using two primers comple-
mentary to the adaptor and restriction site frag-
ments. The fragments are visualized on
denaturing polyacrylamide gels either through
autoradiographic or fluorescence methodologies.
AFLP-PCR is a highly sensitive method for
detecting polymorphisms in DNA. The proce-
dure of this technique is divided into three steps:
- Digestion of total cellular DNA with one or
more restriction enzymes and ligation of
restriction half-site specific adaptors to all
restriction fragments. - Selective amplification of some of these
fragments with two PCR primers that have
corresponding adaptor and restriction site-
specific sequences. - Electrophoretic separation of amplicons on a
gel matrix, followed by visualization of the
band pattern.
(f) Gene-specific PCR
Gene-specific PCR methods are based on the dis-
covery that certain enterotoxin genes are carried
almost exclusively by E. coli that infect indi-
vidual species of warm-blooded mammals inclu-
ding humans, cattle, and swine. E. coli samples
are isolated from water and the DNA extracted.
The samples are amplified and probed with the
genes specific for man and other animals.
The procedure is relatively simple and can be
performed within two working days. The advan-
tages of the gene-specific methods are that they
are highly specific and library independent.
(g) The use of bacteriophages
Bacteriophages (viruses) that infect E. coli and
possibly other closely related coliform bacteria
are called coliphages (Stewart-Pullaro et al.
2006 ). Coliphages were first proposed as indica-
tors of the presence of E. coli bacteria and are
taxonomically very diverse, covering the follow-
ing six virus families: three families of double-
stranded DNA viruses (Myoviridae, Styloviriae,
Podoviridae), two families of single-stranded
DNA phages (Microviridae and Inoviridae), and
one family of single-stranded RNA viruses
(Leviviridae). Coliphages that infect via the host
cell wall of E. coli are called somatic coliphages
(including families Myoviridae, Styloviriae,
Podoviridae, and Microviridae). Male-specific
(also called F+) coliphages (Inoviridae and
Leviviridae) infect by attaching to hair-like
appendages called F-pili protruding from the
host bacterium surface (Fig. 7 .3).
Male-specific coliphages have been studied
extensively as fecal indicators and for water/Fig. 7.3 Pilus linking a male to a female Escherichia coli cell
in conjugation (Image reproduced with permission)