Zika, Dengue and Chikungunya; 3 viruses alike in their symptoms and their transmission by Aedes mosquitoes. Former PCR-based Zika tests were unable to rule out infections with Dengue or Chikungunya. A trioplex real-time RT-PCR assay has now been developed for the detection and differentiation of RNA from these three viruses. This qPCR assay has been approved by the Food and Drug Administration (FDA) for use by the Centre for Disease Control and Prevention (CDC).
The document details the use of a TaqMan® RT-qPCR assay to differentiate Zika RNA from Dengue and Chikungunya using a single sample from either serum, whole blood (EDTA) or cerebrospinal fluid.
“Trioplex Real-Time RT-PCR Assay includes primers and dual-labeled hydrolysis (Taqman®) probes to be used in the in vitro qualitative detection of Zika virus RNA isolated from clinical specimens including serum (from serum separator tubes), whole blood (EDTA), CSF, urine, and amniotic fluid. A reverse transcription step produces cDNA from RNA present in the sample. The probe binds to the target DNA between the two unlabeled PCR primers. For the dengue virusspecific probe, the signal from the fluorescent dye (FAM) on the 5’ end is quenched by BHQ-1 on its 3’ end. For the chikungunya virus-specific probe, the signal from the fluorescent dye (HEX) on the 5’ end is quenched by BHQ-1 on its 3’ end. For the Zika virus-specific probe, the signal from the fluorescent dye (Texas Red [TxRd]) on the 5’end is quenched by BHQ-2 on its 3’ end. During PCR, Taq polymerase extends the unlabeled primers using the template strand as a guide, and when it reaches the probe it cleaves the probe separating the dye from the quencher allowing it to fluoresce. The real-time PCR instrument detects this fluorescence from the unquenched dye. With each cycle of PCR, more probes are cleaved resulting in an increase in fluorescence that is proportional to the amount of target nucleic acid present.”
Full document available here.