Early and Rapid Detection of Denguevirus Serotypes (1-4) by Colorimetric RT-LAMP Amplification Assay

WERBAJH SANTIAGO; MALNERO, C.M; STOLOWICZ, FABIANA; LAROCCA, LUCIANA; CARRILLO CAROLINA; MALIRAT, VIVIANA; VOJNOV, ADRIAN

Buenos Aires

Otro; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Dengue viruses (DENV), a member of the Flaviviridae family, areresponsible 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 economiccosts, mainly due to the lack of approved vaccine or antiviral drugs.Infections with one of the four serotypes of DENV (DENV-1?4) resultin symptoms ranging from an acute, self-limiting febrile illness, dengue fever, to severe dengue haemorrhagic fever or dengue shocksyndrome. The aim of this work is to develop an assay for early andrapid detection of DENV infection during the febrile period. An earlydetection is crucial for proper patient management and prevention of disease spread, especially in the resource-limited rural healthcaresettings where dengue is endemic. Here, a specific, sensitive, androbust prototype colorimetric reverse transcriptase loop-mediatedisothermal amplification (RT-LAMP) assay was developed for detection and differentiation of DENV1-4 serotypes. These techniquesare used as a powerful tool for screening and diagnosis of infectiousdiseases. This Isothermal method, as an alternative to polymerasechain reaction (PCR), require no thermocycling machine and canmostly be performed with reduced time, high throughput, and accurate and reliable results. The reaction was performed in one stepin a single tube by mixing primers, reverse-transcriptase and DNApolymerase together with the tested samples (RNA genome international reference for Dengue serotypes) at 65 ◦C for 60 min, with theaddition visible pH indicator dye prior to amplification. The pH indicator dye change resulting from amplification reactions performedwith 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 reversetranscription-polymerase chain reaction (qRT-PCR).