Detection and subtyping of dengue 1–4 and yellow fever viruses by means of a multiplex RT-nested-PCR using degenerated primers

Differential diagnosis of infections that cause similar diseases and may be active simultaneously in the same geographical areas is greatly needed. Dengue and yellow fever viruses (DENV and YFV) are transmitted by the same species of mosquito and both can cause haemorrhagic fever symptoms. These viruses are active mainly in regions where expensive and sophisticated technologies are not available. Our objective was to develop a simple, reliable and easy-to-perform method to detect and identify these viruses.We slightly modified a generic RT-PCR able to detect the mentioned viruses and other members of this genus: specific primers for each one of these viruses were designed and included in the nested reaction instead of one of the generic ones. The reaction was optimized and viruses are amplified giving rise to bands of different sizes distinguishable in agarose gels.This test is able to detect and identify the four DENVs and YFV to a high level of sensitivity and specificity and can be used with clinical samples. This simple, reliable and easy-to-perform method able to detect and identify dengue 1-4 and YFV can be used in poor endemic countries.