Adaptive Multiplexed RT-PCR Assay for Detection of Chikungunya, Dengue, and Zika Viruses

Abstract

As chikungunya, dengue, and Zika virus infections all present similar symptoms, a multiplexed assay could provide enhanced diagnostic power to clinicians at the point-of-care in low-resource settings: a patient with these symptoms could undergo one test instead of three. This thesis describes the improvement of Adaptive PCR through addition of both a reverse transcription step and multiplexing capability, as well as the development of a multiplexed RT-PCR reaction for simultaneous detection of chikungunya, dengue, and Zika viruses. To implement the reverse transcription step, we selected a molecular beacon sequence to have the greatest slope in intensity vs. temperature at the range best for reverse transcription (45-60°C). The current Adaptive PCR LabVIEW system was programmed to fluorescently detect the correct temperature range and hold the system’s temperature as near to that point as possible. Primers and probes were optimized for each of the viral RNA genomes to specifically detect only one of the three similar viruses. A different fluorophore was conjugated to each of the probes to allow detection on different channels. The multiplexed assay gave a limit of detection of 5 RNA copies/reaction for Zika and chikungunya and 50 RNA copies/reaction for dengue, all of which are at or just above the average clinical viral load of patients and therefore extremely useful in diagnosis. In addition, none of the negative controls amplified, showing high sensitivity and specificity. Portable multiplexed Adaptive RT-PCR will promote proper diagnoses of chikungunya, dengue, and Zika viruses at the point-of-care, allowing for proper treatment.