New Approaches for the Identification of Drug Targets in Protozoan Parasites - International Review of Cell and Molecular Biology - page 364

most sensitive method” detecting “one copy” of a gene. This may be true
under ideal conditions. One has to consider, however, that only a minor
part of the DNA extracted from wells of a culture plate is analyzed by
one PCR reaction. For example, if DNA from a well is eluted in 50 µl
and if it contains 10 copies of the gene to be detected, the probability
for 5 µl to contain 1 copy is according to the Poisson distribution 0.367
(1/e), the same as for 0 copies. To ensure a correct detection of a small
number of gene copies, the PCR reactions must be upscaled by a factor
of 10. On the other hand, 10 T. gondii or N. caninum tachyzoites express-
ing Escherichia coli beta-galactosidase can be detected in 96-well plates
in the presence of host cells by a mere increase of the incubation time
( McFadden et al., 1997 ; own unpublished results). A similar sensitivity
is described for T. gondii expressing yellow fluorescent protein ( Gub-
bels et al., 2003 ). The use of fluorescence has, however, the following
disadvantages: i) the sensitivity cannot be extended by a mere increase
of incubation time; ii) care must be taken to minimize autofluorescence
by host cells, medium compounds, drug residues, etc. In some instances,
cells have to be washed and overlaid with a convenient buffer. A clear
advantage of strains expressing fluorescent proteins is that they allow a
continuous monitoring of parasite proliferation under nondestructive
conditions, even in the presence of host cells with no or little back-
ground.
In order to quantify only the viable parasites (as opposed to the overall
number of parasites), vitality assays can be employed. Gene expression
occurs only in viable cells, and this may qualify assays based on the incor-
poration of nucleic acid precursors and assays based on the use of trans-
genic parasites as a kind of vitality assay. The quantification of mRNA of
a parasite-specific gene by quantitative RT-PCR ( Strohbusch et al., 2008 )
has been a step in this direction since mRNA has a shorter half life than
DNA. The half lives of nucleic acids and proteins are, however, much lon-
ger than the half lives of the intermediary metabolites detected in vitality
assays that quantify actively metabolizing cells, only. They are based on the
detection of intermediate metabolites with a rapid turnover, such as ATP
using the luciferin-luciferase assay ( Chen and Cushion, 1994 ), or NADH
which is quantified with tetrazolium salts ( Wright et al., 1992 ) or with
resazurin ( Bénéré et al., 2007 ). When employed with G. lamblia , the resa-
zurin or “Alamar Blue” assay allows a rapid and reliable quantification of
trophozoites grown in microtiter plates, but care must be taken to remove
the reducing medium by repeated washes with prewarmed PBS since