Diagnóstico molecular de dengue e zika por transcrição reversa seguida da amplificação isotérmica mediada por loop (RT-LAMP) em dispositivo a base de papel

Abstract

Dengue and zika are viral infectious diseases occurring in countries with a tropical and subtropical climate in which around 3.6 billion people live. It is estimated that in 50 to 100 million new cases of dengue occur annually, generating economic, social and public health impacts. Dengue and zika have usually been diagnosed by serological methods which are generally of low confidence, as they can generate false-positive results, which are related to the presence of antibodies produced against previous infections of the virus. The molecular methods are more accurate, however the molecular method most commonly used (PCR) requires a long time of reaction and requires sophisticated instrumentation and high cost, making point of care applications difficult,, especially in developing countries. This work presents the development of a molecular diagnostic methodology in a paper-based platform that allowed the detection of the virus through the reverse transcription -loop mediated isothermal amplification (RT-LAMP). The reactions were carried out on 6 mm diameter FTA paper discs, confined in a multi-layered polyester-toner device, incubated at 65 ° C for 45 minutes in a dry bath and then performed visual detection using the SYBR Green intercalator. Positive reactions were identified by the green fluorescence emitted after the addition of the intercalator. The results were recorded through the capture of the images by a photodocumentator and/or by smartphone and later analyzed by the software ImageJ, allowing the comparison between negative and positive reactions. The methodology developed for the detection of the virus by RT-LAMP in paper substrate was sensitive, being able to detect the virus in initial concentrations of 0.1 pg μL -1 of RNA in the master mixture. In addition, it was possible to detect the virus directly in complex samples (serum of infected patients) without the need of previous viral RNA extraction step. Elimination of the RNA extraction step together with the visual detection on the paper produce the final result in 46 minutes. The results demonstrated that the detection of the virus by RT-LAMP in paper substrates is a valuable tool for the molecular diagnosis of infectious diseases, presenting great potential for point-of-care applications for both diagnostics and epidemiological studies, especially in developing countries.