Multiplex real-time RT-PCR for detection and distinction of Spondweni and Zika virus

Zika (ZIKV) and Spondweni viruses (SPOV) are closely related mosquito borne flaviviruses in the Spondweni serogroup. The co-circulation and similar disease presentation following ZIKV and SPOV infection necessitates the development of a diagnostic tool for their simultaneous detection and distinction. We developed a one-step multiplex real-time RT-PCR (ZIKSPOV) to detect and distinguish between SPOV and ZIKV by utilizing a single primer set combined with virus specific hydrolysis probes. The ZIKSPOV assay was compared to published virus specific real-time RT-PCR assays and the limit of detection was comparable. The SPOV reference strain AR94 was detectable to 0.001 TCID₅₀ per PCR reaction, while African lineage ZIKV (MR 766) was detectable to 0.002 TCID₅₀ per reaction and Asian lineage ZIKV (H/PF/2013) to 0.05 TCID₅₀ per reaction. The ZIKSPOV assay did not detect other flaviviruses, indicative of its specificity for Spondweni serogroup. The ZIKSPOV assay is a useful addition to arbovirus diagnostic and surveillance tools in areas where ZIKV and SPOV are expected to co-circulate. Further evaluation is required to demonstrate the application of the assay for detection of ZIKV and SPOV in mosquito and human clinical samples.