Zika virus (ZIKV) has been a cause of worldwide concern, especially after findings that microcephaly could be caused by a mutation of the ancestral strain. There are two major lineages of ZIKV, African and Asian, and evidence has been encountered that both lineages have evolved into mutated sequences. ZIKV lineages have been studied in an effort to understand their impact on brain development. A study concluded that African isolates grow faster, are more lethal, and cause more virulent brain damage than Asian ZIKV. It has been difficult to achieve a methodology that can differentiate between African and Asian ZIKV, due to high genetic similarity and antigen cross-reactivity. In our approach, we present a detection methodology able to differentiate African ZIKV and Puerto Rican ZIKV, an isolate of Asian ZIKV lineage. To achieve differentiation, an electrochemical biosensor was designed and African ZIKV RNA was amplified using Nucleic Acid Sequence Based Amplification (NASBA), an isothermal amplification technique. After analyzing five African ZIKV isolates, a universal fragment was found that can also be used to discriminate against Puerto Rican ZIKV. This detection method uses a 4-way junction (4WJ) structure, formed by immobilizing a universal stem-loop probe on the gold substrate via a thiol bond, along with two adaptor strands, m and f. The signal response is monitored by attaching methylene blue (redox marker) to the m-strand and measuring the presence of the target using square wave voltammetry. Optimal hybridization signals were obtained from synthetic samples at 10 minutes, with a peak value of 3.2 μA/cm2and a limit of detection of 0.95 nM. This methodology was tested for NASBA products of African ZIKV, Puerto Rican ZIKV, and Dengue, which were amplified using African Zika primers. The biosensor presented current densities of -4.20 ± 0.03 μA/cm2, -0.93 ± 0.07 μA/cm2, and -0.10 ± 0.12 µA/cm2for African ZIKV, Puerto Rican ZIKV, and Dengue respectively. These results show promise as a way to differentiate between African ZIKV and Asian ZIKV’s derivatives. This electrochemical method of detection is also low-cost, efficient and would be ideal for point-of-care applications worldwide.

Differentiation between ZIKA lineages using electrochemical biosensors based on nucleic acid detection

2019 Spring National Meeting

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