The recent outbreak of Zika virus (ZIKV) in the Americas led the World Health Organization to declare ZIKV as a Public Health Emergency of International Concern, due to correlation with neurological complications, such as microcephaly and Guillain-Barre Syndrome. Currently, ZIKV detection methods have cross-reactivity with other flaviviruses or use expensive and time-consuming techniques. Thus, the development of a cost-efficient, selective and rapid method to detect ZIKV is important. Here, we demonstrated a new methodology that relies on highly selective electrochemical detection/quantification of RNA from ZIKV circulating in the Americas. In our approach, a universal stem-loop probe (immobilized in a gold substrate) indirectly interacts with a fragment of viral RNA forming a four-way junction (4WJ) structure with the help of two adaptor strands (m and f). Strand-m is equipped with a redox marker (methylene blue-MeB), so that only in the presence of a specific targeted RNA fragment, MeB is brought to the electrode’s surface, producing the current changes as a signal output. The signal can be conveniently detected by square wave voltammetry after as little as 10 min of hybridization time. A target fragment of ZIKV RNA (159 nt) obtained using Nucleic Acid Sequence Based Amplification (NASBA) was detected without the need for the amplicon purification. Control experiments were performed in the absence of the target and/or strand-f, and a negligible signal was observed. Amplification time and starting concentration of viral RNA for NASBA was evaluated. The highest current density of 4.9 ± 0.3 µA/cm2 was achieved for the amplicon after 45 min of NASBA reaction. Linear correlation between the signal and the logarithm of the RNA concentration was observed in the range of 5-500 fg/µL, with the limit of detection of 1.11 fg/µL (or 1.8 x 105 ZIKV RNA copies/mL), which is lower than usually found in clinical samples (7 x 105 copies/mL). The selectivity was analyzed using RNA from West Nile virus and serotypes 1-4 of dengue virus amplified with ZIKV primers. A significant signal was observed only for the ZIKV amplicon (4.7 ± 0.7 µA/cm2), while the other samples presented signal lower than 0.2 ± 0.1 µA/cm2, similar to the blank value. The proposed methodology is promising for point-of-care diagnostics of ZIKV infections, since the viral RNA was successful detected with high selectivity and optimal detection limit in less than 1 hour using NASBA and 4WJ platform.

Rapid and selective detection of Zika virus circulating in the Americas using electrochemical RNA-sensor along with NASBA

2019 Spring National Meeting

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