Programmable low-cost DNA-based platform for viral RNA detection

Lifeng Zhou; Arun Richard Chandrasekaran; Jibin Abraham Punnoose; Gaston Bonenfant; Stephon Charles; Oksana Levchenko; Pheonah Badu; Cassandra Cavaliere; Cara T. Pager; Ken Halvorsen

In addition to possible misdiagnosis between different viruses, there is an additional challenge in determining the specific strain of a virus. For example, in Latin America four different DENV serotypes are known to be present and co-circulate, where misdiagnosis of the infecting strain can have significant implications for treatment options. 35 Thus being able to accurately identify a circulating strain of virus broadly impacts medical care, surveillance and vector control. 36 ZIKV was first identified in Uganda in 1947 before spreading to Asia and the Americas, and ZIKV strains (classified within African or Asian lineage) share significant sequence homology. 37 To investigate if our assay can distinguish between the Asian and African lineages, we tested our nanoswitches against two ZIKV strains which have a ~89% sequence homology, namely the FSS13025 isolated from Cambodia and the MR766 strain isolated from Uganda. In designing the ZIKV strain-specific nanoswitches, we identified five target regions that each have a 5-6 nucleotide difference ( Fig. 2C and S12) . To achieve better discernment of the detection signal, the nanoswitches for the Uganda strain were designed to form a smaller loop-size than those designed for Cambodia. Next, a human hepatocellular carcinoma cell line (Huh7) was infected with either the Cambodian or Ugandan ZIKV strain. Infected cells were processed to extract total RNA, which was then fragmented and incubated with nanoswitches to probe for viral RNA from either the ZIKV Cambodia or ZIKV Uganda infected cells. The results showed that our assay was able to discriminate between two strains of the same virus even with high genetic similarities (Fig. 2D, S12) .