Development of a colorimetric RT-LAMP amplification assay adapted to an early and easy detection of Zika, Chikungunya and the four serotypes of Dengue virus

WERBAJH S; CRISTIAN MALNERO; STOLOWICS FG; L. LAROCCA; ALICIA MISTCHENKO; VIVIANA MALIRAT; CAROLINA CARRILLO; VOJNOV A

Congreso; LXIV Reunión anual de la Sociedad Argentina de Investigaciones Clínicas; 2019

The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. These arboviruses are transmitted by mosquitoes, and cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid and specific discrimination of these three viruses in patient samples is needed. The aim of this work was to develop a simplified test for DENV serotypes, CHIKV and ZIKAV detection in early stages of infection. Methods & Materials: We developed a colorimetric reverse transcriptase loop-mediated isothermal amplification assay (COLOR-RT-LAMP), an alternative method of polymerase chain reaction (PCR) that, as it works at a fixed temperature, not require thermocyclers and, also, can be performed in short times, showing accurate and reliable results. A single-tube was developed with specific primers, RT and DNA polymerase together with the tested samples (RNA genomes references). Ranges of temperature, pH, Mg and time were evaluated. The readout of the test was defined as a color change -visible to a naked eye-, by the addition of neutral red dye, or HNB dye (Hydroxy naphthol blue) prior to the amplification; these dyes changes its color as a result of the amplification. The sensitivity and specificity of the RT-LAMP were evaluated.Results: Here we described the achievement of an early and easy method to detect and diferentiate DENV serotypes, ZIKAV and CHIKV. The results showed that our ColorRT-LAMP test is highly sensitive and specific. No cross-reactivity was observed with all other three closely related arboviruses. Conclusion: These COLOR-RT-LAMP method used in our study is specific, sensitive, and suitable for further investigation as a useful alternative to the current methods used for clinical diagnosis and diferentiated of DENV1-4, ZIKAV and CHIKV, especially in hospitals and laboratories that lack sophisticated diagnostic systems.