Recently, Schneider et al. (2013) have reported a novel VNTR-based
(Variable-Number-Tandem-Repeat) molecular screening tool for detecting
Wolbachia infections in tsetse flies and showed that their infections
in Glossina spp. can escape the standard PCR screening methods
by ‘hiding’ as low-titer infections below the detection threshold. So, it
is possible that not all infections were detected in the current work due to the low titer infections of Wolbachia. However, another method
using real-time quantitative PCRwas applied to this study for confirmation
ofWolbachia infections. Real-timequantitative PCR requires the absolute
quantification of ftsZ gene which is a single copy per genome in
Wolbachia, so DNA standard with known concentration was required
and the host single copy genewas also quantified in order to allow normalization
for making a comparison among species. The real-time PCR
does not detect PCR products butmeasures fluorescence that is released
from a reporter dye attached to the probe. Thus, threshold cycles, in
which the fluorescence begins to increase from the background level,
are measured. The serial dilution of standards produced a reliable standard
curve that decreases in any differences in the binding efficiencies
of primers and probe with DNA samples. A specific probewas used for
detecting the ftsZ gene and the host gene detection from all DNA samples.
In addition to P. perpusilla, all planthopper sp. examined here indicated
significantly higher Wolbachia densities than leafhopper sp. This
result may relate to the importance of pests as most planthoppers are
major pests in rice agriculture.
Recently, Schneider et al. (2013) have reported a novel VNTR-based(Variable-Number-Tandem-Repeat) molecular screening tool for detectingWolbachia infections in tsetse flies and showed that their infectionsin Glossina spp. can escape the standard PCR screening methodsby ‘hiding’ as low-titer infections below the detection threshold. So, itis possible that not all infections were detected in the current work due to the low titer infections of Wolbachia. However, another methodusing real-time quantitative PCRwas applied to this study for confirmationofWolbachia infections. Real-timequantitative PCR requires the absolutequantification of ftsZ gene which is a single copy per genome inWolbachia, so DNA standard with known concentration was requiredand the host single copy genewas also quantified in order to allow normalizationfor making a comparison among species. The real-time PCRdoes not detect PCR products butmeasures fluorescence that is releasedfrom a reporter dye attached to the probe. Thus, threshold cycles, inwhich the fluorescence begins to increase from the background level,are measured. The serial dilution of standards produced a reliable standardcurve that decreases in any differences in the binding efficienciesof primers and probe with DNA samples. A specific probewas used fordetecting the ftsZ gene and the host gene detection from all DNA samples.In addition to P. perpusilla, all planthopper sp. examined here indicatedsignificantly higher Wolbachia densities than leafhopper sp. Thisresult may relate to the importance of pests as most planthoppers aremajor pests in rice agriculture.
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