Biology Reference
In-Depth Information
Protozoan targets
Numerous studies have investigated various DNA sequences of protozoa
as potential molecular targets to confirm their presence in food and water
samples. The use of PCR to confirm
Giardia
and
Cyptosporidium
presence
has been employed for over a decade; however, they still are used only as
confirmatory and not as stand-alone monitoring tests. As with bacteria, the
small subunit rRNA gene (18S rRNA gene) has been the focus of several
studies with both
Cyptosporidium
and
Giardia
.
67-69
Although the method is
the most sensitive for species identification, it still needs to be coupled with
sample concentration methods because of the low level of parasitic DNA.
Other genes have also been studied, such as the heat shock (
hsp70
) for
Cryptosporidium parvum
70
and
Giardia
,
67
the elongation factor 1a (
Giardia
)
and the triose phosphate isomerise gene (
tpi
) (
Giardia
).
71
As all species of
Cryptosporidium
oocysts respond to a heat shock by producing the protein
Hsp70,
72
the mRNA gene coding for hsp70 can also be used as a viability
marker. However, in the view of some water companies, this method has
not been sufficiently validated for being of practical use.
73
More recently, Wang et al.
74
developed a microarray containing a large
variety of targets to simultaneously detect multiple protozoan parasites
in samples. They used the previously tested conserved target genes such
as rRNA and
hsp
70 to identify genus or species but also included highly
variable genes such as cysteine protease 1 (
cp1
) for
Entamoeba
,
c4
(species
specific open reading frame) for
Giardia
and
Crytosporidium
oocyst wall
protein (
cowp
), polythreonine-rich glycoprotein (
ptg
), the thrombospondin-
related anonymous protein 2 gene (
TRAP-C2
), and protein 23 (
p23
) for
Cyptosporidium
to be able to identify down to the species, assemblage, or
genotype level. As few as five trophozoitres of
Giardia
could be accurately
detected by this method.
74
It remains to be determined if this method can
be incorporated into routine testing of environmental water samples.
8.2.3.3. Quantitative and real-time quantitative PCR
The PCR reaction generates copies of the target sequence exponentially
initially. Because of inhibitors of the polymerase reaction found in the
sample, reagent limitation, accumulation of pyrophosphate molecules, and
self-annealing of the accumulating product, the PCR reaction eventually
ceases to amplify target sequence at an exponential rate and a “plateau
effect” occurs, making the endpoint quantification of PCR products unre-
liable. Only during the exponential phase of the PCR reaction is it pos-
sible to extrapolate back to determine the starting quantity of the target
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