OBJECTIVE: The re-emergence and spreading of tropical viruses to new areas raised worldwide a wave of concern. To treat patients in an early stage and prevent diffusion of the outbreak, an early diagnosis, and thus fast and adequate detection, is needed. To this aim, a multiplex reverse transcription real-time polymerase chain reaction TaqMan® method was designed to detect universally Zika and chikungunya viruses.

DESIGN: Two methods, targeting different genome segments, were selected from literature for each virus, adapted for high genome coverage and joined into a four-plex assay which was thoroughly in-house validated. The SCREENED tool was used to evaluate the sequence coverage of the method.

RESULTS: The full validation approach showed that the new four-plex method allows specific and sensitive identification and discrimination of Zika and chikungunya in routine samples. The combination of two targets per virus allowing almost 100% coverage of about 500 genomes was shown for the first time.

CONCLUSION: PCR being a reliable user-friendly technique, applicable in remote areas, such multiplex methods enable early and efficient diagnosis leading to rapid treatment and effective confinement in outbreak cases as well as being an aid for surveillance and the full validation permits easy method-transfer allowing worldwide harmonization.