Testes moleculares baseados em amplificação isotérmica mediada por Loop para detecção de arbovírus em microdispositivos descartáveis

Abstract

Arboviruses such as dengue virus (DENV) and zika virus (ZIKV) are transmitted to humans through the bite of Aedes mosquitoes. Infections with arboviruses presents similar symptoms and commonly cross reactions. The incidence of these viruses emerges in general in resource limited settings, where sophisticated laboratory infrastructure and trained professionals to make an accurate, reliable and differential diagnosis is not available. Hence, a diagnostic method that are simple, cost effective, reliable and point of care applicable is necessary. In this study we develop assays for the detection of arbovirus in real sample from infected patients by a reverse transcription reaction followed by a loop-mediated isothermal amplification (RT-LAMP) in a polyester toner (PeT) disposable microdevice. The reactions were thermally controlled with a thermoblock and in 10 min it was possible to detect the ZIKV and in 15 min it was possible to detect the DENV. At the end of the incubation time, detection was

performed by electrophoresis (off-chip detection) or a DNA fluorescent dye was added for on- chip detection (on-chip detection). Our results demonstrated that it is possible to detect DENV

and ZIKV through an RT-LAMP assay directly on samples from infected patients, without a previous RNA extraction step. The success of RT-LAMP was confirmed in reactions initiated with 200 copies of RNA μL-1

for dengue and 0.2 copies of RNA μL-1

for Zika virus. RT-LAMP performed on the PeT microdevice is a simple and inexpensive method that allows for rapid detection of dengue virus with high reliability and great potential for point-of-care applications.