Early and rapid detection of dengue virus serotypes 1?4 by colorimetric RT-LAMP amplification assay

WERBAJ S; MALNERO C; STOLOWICZ FG; LAROCCA L; CARRILLO C; MARILAT V; VOJNOV AA

Ciudad autonoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencia 2017; 2017

Sociedades Científicas Argentinas

Dengue viruses (DENV), a member of the Flaviviridae family, are responsible for one of the most important emerging viral diseases. This mosquito-borne disease has a great impact in tropical and subtropical areas of the world in terms of illness, mortality and economic costs, mainly due to the lack of approved vaccine or antiviral drugs. Infections with one of the four serotypes of DENV (DENV-1?4) result in symptoms ranging from an acute, self-limiting febrile illness, dengue fever, to severe dengue haemorrhagic fever or dengue shock syndrome. The aim of this work is to develop an assay for early and rapid detection of DENV infection during the febrile period. An early detection is crucial for proper patient management and prevention of disease spread, especially in the resource-limited rural healthcare settings where dengue is endemic. Here, a specific, sensitive, and robust prototype colorimetric reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection and differentiation of DENV1-4 serotypes. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. This Isothermal method, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results. The reaction was performed in one step in a single tube by mixing primers, reverse-transcriptase and DNA polymerase together with the tested samples (RNA genome international reference for Dengue serotypes) at 65 ◦C for 60 min, with the addition visible pH indicator dye prior to amplification. The pH indicator dye change resulting from amplification reactions performed with minimal buffering, by eye without a need for instrumentation. The result showed no cross reactivity to other closely related arbovirus (Zika). Our assay is more sensitive than quantitative reverse transcription-polymerase chain reaction (qRT-PCR).