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RNA AS A FEASIBLE MARKER OF TRYPANOSOMA CRUZI VIABILITY DURING THE PARASITE INTERACTION WITH THE TRIATOMINE VECTOR RHODNIUS PROLIXUS (HEMIPTERA, TRIATOMINAE)
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Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Universidade Federal Fluminense. Niterói, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil / Universidade Federal Fluminense. Niterói, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Bioquímica e Fisiologia de Insetos. Rio de Janeiro, RJ, Brasil.
Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ, Brasil.
Abstract
A recurring question concerning Trypanosoma cruzi DNA detection/quantification is related
to the fact that DNA amplification, by itself, does not differentiate between viable or dead
parasites. On the other hand, RNA can be considered a potential molecular marker of patho gens viability. Herein, we developed a quantitative real-time PCR with reverse Transcription
(RT-qPCR) to quantify viable T. cruzi in artificially infected Rhodnius prolixus whilst evaluat ing differences between DNA and mRNA quantification along the insect midgut during 5, 9,
15 and 29 days after feeding. The RT-qPCR presented an improved performance with line arities ranging from 107 to 102 parasites equivalents and 3 to 0.0032 intestine unit equiva lents, and efficiencies of 100.3% and 102.8% for both T. cruzi and triatomine targets,
respectively. Comparing both RT-qPCR and qPCR, we confirmed that RNA is faster
degraded, no longer being detected at day 1 after parasite lysis, while DNA detection was
stable, with no decrease in parasite load over the days, even after parasite lysis. We also
observed statistical differences between the quantification of the parasite load by DNA and
by RNA on day 15 after feeding of experimentally infected R. prolixus. When assessing dif ferent portions of the digestive tract, by RT-qPCR, we could detect a statistically significant
reduction in the parasite amount in the anterior midgut. Oppositely, there was a statistically
significant increase of the parasite load in the hindgut. In conclusion, for this study parasite’s
viability in R. prolixus digestive tract were assessed targeting T. cruzi mRNA. In addition, dif ferences between DNA and RNA detection observed herein, raise the possibility that RNA is
a potential molecular viability marker, which could contribute to understanding the dynamics
of the parasite infection in invertebrate hosts.
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