One-step Pentaplex Real-time Polymerase Chain Reaction Assay for Detection of Zika, Dengue, Chikungunya, West Nile Viruses and a Human Housekeeping Gene

Background: Recent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections.

Objectives: We established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction.

Study Design: Specific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 10 GEQ/Rn for ZIKV, 2.2 × 10 GEQ/Rn for DENV-1, 9.4 × 10 GEQ/Rn for DENV-2, 2.3 × 10 GEQ/Rn for DENV-3, 1.4 × 10 GEQ/Rn for DENV-4, 2.7 × 10 GEQ/Rn for CHIKV, and 1.05 × 10 GEQ/Rn for WNV.

Conclusions: The CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction.