Biology Reference
In-Depth Information
the growth of the three bloom formers. With the help of real-time PCR of
mcyA
and phycocyanin
markers it was possible to survey the fl uctuations in the populations of MC-producing and non-
MC-producing
Planktothrix agardhii
in a eutrophic French Lake. The proportion of MC-producing
genotype fl uctuated between 30-80% of the poupulation (Briand
et al
., 2008).
vii) Taq nuclease assay or 5'-nuclease PCR
:
TaqMan
(TM) PCR or
Ta q
nuclease assay was fi rst developed
to enumerate specifi c genotypes of picocyanobacteria (Becker
et al
., 2000). This was extended for
simultaneous monitoring of two microbes (
E
.
coli
and MC-producing strains of
Microcystis
) by conducting
PCR reaction sequence of specifi c probe dually labelled with fl uorescent dye that is also known as
TaqMan
probe (Foulds
et al
., 2002). The rate of exponential accumulation of the amplifi con is monitored
by the hydrolysis of TM probe in which it generates a fl uorescent signal during the amplifi cation
process. To detect
E
.
coli
lacZ
TM probe with a sequence of 5'-ATTCGCCATTCAGGCTGCGCAA-3'
and for the identification of a MC TM probe having a sequence of 5'-TTAAATCGGAAAT
TATCCCAGAAAATGCCGT-3' were selected. These dual labelled probes were modified
with 5'-6-carboxyfluorescein reporter dye and a 3' 6-carboxy N, N, N', N' tetramethylrho-
damine quencher dye. LacZ primers were employed to amplify a region of
lacZ
gene in
E
.
coli
(180 bp amplifi con) for the identifi cation and enumeration of
E
.
coli
cells contaminating drinking
water. In a similar manner, a region of
mcyA
gene from MC synthetase operon (1220 bp amplifi con)
was identifi ed to be specifi c for toxin-producing strains of
M
.
aeruginosa
. 5'-Nuclease PCR reactions
were performed in which a standard curve based on pre-determined cell or DNA concentrations
was compared to assay the threshold cycle of unknown samples. The concentration of microbes
in water samples was quantifi ed by knowing standard units of gene copies of
lacZ
gene and
mcyA
gene µL
-1
. Using the above methods, it was possible to detect three copies of target samples within
2 h after DNA extraction from both microbes (Foulds
et al
., 2002).
Seasonal development of bloom populations of
Microcystis
was monitored along with toxic
MC-producing genotype in Lake Wannsee (Berlin, Germany).
Ta q
nuclease assay was performed
for two gene regions, i.e.
PC
-
IGS
sequence for the estimation of total population and
mcyB
gene of
mcy
gene cluster. The development of threshold cycle of PCR enabled to infer the number of cells.
These showed a correlation with microscopically determined cell numbers from logirthmic cultures.
Identical amplifi cation effi ciencies for both genes were detected in all 10 strains of
Microcystis
cultures
examined. The application of
Ta q
nuclease assay for fi eld samples revealed that the mean number
of
mcy
genotype is stable from winter to summer and in Lake Wannsee its cell numbers could be
enumerated. Further, the
mcy
genotypes formed a smaller part of the PC genotypes ranging from 1
to 38% and a correlation of 1:1 between cell number and the two selected markers existed. A parallel
relationship between cell numbers estimated through inverted microscope counts and
Ta q
nuclease
assay was also established (Kurmayer and Kutzenberger, 2003).
VII. OCCURRENCE OF HARMFUL ALGAL BLOOMS (HABs)
Signifi cant human health risk from HABs has been predicted on the basis of worldwide surveys on
mass development of toxic cyanobacteria in potable and recreational waters. The percentage of toxic
samples accounted for 60% of the blooms formed world over. In general, hepatotoxin producers are
more common than the neurotoxin-producing ones. However, severe animal poisonings have been
reported from North America, Europe and Australia due to neurotoxins rather than by hepatotoxins.
Blooms producing CYN have been reported from Australia, Hungary, Japan, Israel and Germany.