Padronização da RT-PCR Duplex, Multiplex e Nested para detecção do vírus zika, dengue e chikungunya

Abstract

Zika (ZIKV), Dengue (DENV) and Chikungunya (CHIKV) viruses are the most prevalent arboviruses in the Americas. They share the same vector, the Aedes aegypti mosquito, overlap in their endemic areas and have similar clinical symptoms, especially in the initial stages of the infection. In recent years, these arboviruses have increased their area of endemicity and it has been considered a major public health problem. This reinforced the need for the development of laboratory tests to accurately confirm infections by these viruses. These tests should be rapid, specific, efficient, cost-effective and easy to perform, allowing identification of viruses in patients presenting the disease symptoms. This work proposes the standardization of protocols for the identification of the Alphavirus and Flavivirus genera, as well as the simultaneous detection of ZIKV, DENV and CHIKV viruses in a single reaction and differentiation of DENV serotypes. For this, nine pairs of primers already described in the literature which were sensitive and specific to the virus target sequence were used. Three different RT-PCR modalities were used to achieve this purpose, Duplex RT-PCR, Multiplex RT-PCR and Nested RT-PCR. In Duplex RT-PCR reaction, it was only possible to observe the product amplified by primer pair FG1 / FG2, showing that primer pair cM3W / M2W was not as sensitive. In Multiplex RT-PCR reaction, it was only possible to observe the products amplified by DEN-F / DEN-CR and ZIKVEN-F / ZIKVEN-R primers, but CHIKF / CHIKR product was not observed, however, when the reaction was performed separately it was possible to observe all products amplified by each of the primers. In Nested RT-PCR reaction, it was possible to observe only the products amplified by the primers DEN-F / DEN-CR, DEN-1 and DEN-2. These results show that it is still necessary to optimize the primers used and the reactions to increase the sensitivity of the tests performed in this study so that a test can be standardized for the detection of the three arboviruses described herein.