Development of a colorimetric RT-LAMP amplification assay adapted to an early and easy detection of Dengue virus

Abstract

Background: Dengue viruses (DENV) produce one of the most important emerging viral diseases around the world. This mosquito-borne disease has the greater impact in tropical/subtropical areas, causing symptoms ranging from acute, self-limiting febrile illness, dengue fever, to severe dengue hemorrhagic fever to dengue shock syndrome. The infection is caused by 4 distinct, but closely related, serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). There are neither specific antiviral drugs nor approved vaccines; then, an early detection is a crucial step for proper patient management, lowing fatality rates, and for preventing the spread of the disease. The aim of this work was to develop a simplified test for DENV detection in early stages (during the febrile period of the infection), identifying the serotype involved. Methods & Materials: We developed and evaluated a colorimetric reverse transcriptase loop-mediated isothermal amplification assay (ColorRT- 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. The complete reaction was performed in one step in a single tube by mixing primers, reverse-transcriptase and DNA polymerase together with the tested samples (RNA genomes from the international references of Dengue serotypes). Ranges of temperature and time were evaluated, selecting 65 ∘C for 60 min as the best reaction options. The read out of the test was defined as a color change -visible to a naked eye-, by the addition of a visible pH indicator dye, the neutral red dye, prior to the amplification; this pH indicator changes its color as a result of the amplification performed under minimal buffering conditions. Results: Here we described the achievement of an early and easy DENV detection test. The results showed that our ColorRT- LAMP test is highly sensitive, more than quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and highly specific, differentiating between the four serotypes and not showing cross reaction with another closely related arbovirus (Zika). Conclusion: Here we described the development of a simple ColorRT- LAMP test that could be used as a powerful tool for an early, easy, and sensible detection of Dengue disease.