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

Further applying our technique to detect ZIKV RNA in biological samples, we either mock-infected or infected Huh7 cells with the Cambodia ZIKV strain at a multiplicity of infection of 1 and extracted RNA from the ZIKV infected cells at 1-, 2-and 3-days post-infection. 38 The nanoswitch assay detected ZIKV viral RNA from the infected cells but not the mock infected cells (Figs. 3A, 3B & S13 ). Our detection result shows that the copies of ZIKV RNA within infected cells steadily increased upon the infection and plateaued at 2-and 3-days post-infection (Fig. 3C) . These data demonstrate that our assay can detect ZIKV RNA in infected cell lines, and in contrast to typical RT-PCR assays without amplification of the viral RNA. Moving toward clinical applications, we aimed to demonstrate detection of relevant levels of ZIKV RNA from biological fluids. ZIKV is present in the serum, urine, and other body fluids of infected patients. 39 The viral loads can vary dramatically between individuals, body fluid, and post infection time, 6, 7 but are frequently in the subfemtomolar to femtomolar range, with ZIKV in human urine reported as high as 220 × 10 6 copies/ml (365 fM). 33 While our nanoswitch sensitivity for in vitro transcribed viral RNA in buffer approaches clinically relevant concentrations, detection from body fluids is further challenged by varying viral loads and by body fluids that can reduce the performance of the nanoswitches due to physiological conditions and nuclease activity. 26, 40 To overcome these potential difficulties, we investigated two independent solutions: 1) adding a preprocessing step to extract RNA from body fluids such as urine, or 2) adding an isothermal pre-amplification step. In the first approach, we spiked a clinically-relevant amount of in vitro transcribed ZIKV RNA into human urine and processed viral RNA extraction using a commercial RNA extraction kit. We then mixed the extracted RNA with the nanoswitches and demonstrated non-enzymatic, clinical level detection of the RNA at 0.28 pM (Fig. 4A-B & S14) . In the second approach, we demonstrated that our detection can be coupled with other amplification approaches such as nucleic acid sequence-based amplification (NASBA). 41,42 NASBA combines multiple enzymes and primers to achieve RNA amplification in a one-pot isothermal reaction (Fig. S15A) . First, we showed feasibility of the amplification of ZIKV RNA by NASBA in water, followed by nanoswitch detection (Fig. S15) . To mimic clinical samples, we spiked infectious ZIKV particles into either PBS or 10% human urine at clinical-levels (1.49 fM to 0.03 fM). From these samples our assay detected ZIKV RNA in ~5 hours (Fig. 4C-E and S15) . We went one step further and showed that our assay can be performed using a commercially available bufferless gel cartridge (ThermoFisher E-gel) and imaged on a small and potentially portable gel reader (Fig. S16) . With the help of NASBA amplification, the detection ability of our method has about 10,000-fold increase, from ~350 fM (Fig. 1G) to 0.03 fM (Fig. 4D , E) and the detection time was reduced from ~13 hours to ~5 hours (Fig. S17) .