Development of a TaqMan real time reverse transcriptase PCR assay for detection of Zika, Yellow Fever and Dengue-4 viruses simultaneously in a single reaction

Zika, yellow fever and dengue viruses are three important flaviviruses that are epidemic and endemic in Central Africa, Middle and South America, Southeast Asia and Pacific Islands. Since these three viral diseases present with similar symptoms and there is a possibility of multiple flaviviruses co-infection, it is important to develop an effective and quick diagnostic tool. Here, we designed and validated a one-step triplex real time reverse transcription polymerase chain reaction (RT-PCR) using TaqMan probes targeting NS5 region for detecting zika virus (ZIKV), dengue virus-4 (DENV-4) and yellow fever virus (YFV). The assay was highly specific for the target viral RNA corresponding to ZIKV, DENV-4 and YFV strains. The sensitivity of the assay was compared to one-step, RT-PCR, it showed a 100-fold higher sensitivity than RT-PCR with ZIKV and DENV and 10-fold higher sensitivity with YFV. In conclusion, the one-step, triplex, TaqMan, real time RT-PCR assay identified and distinguished ZIKV, DENV-4 and YFV by targeting single gene.   Key words: Zika, yellow fever, dengue, multiplex real time reverse transcription-polymerase chain reaction (RT-PCR).